Crystal Packing Reveals a Potential Autoinhibited KRAS Dimer Interface and a Strategy for Small-Molecule Inhibition of RAS Signaling.

KRAS GTPases harbor oncogenic mutations in more than 25% of human tumors. KRAS is considered to be largely undruggable due to the lack of a suitable small-molecule binding site. Here, we report a unique crystal structure of His-tagged KRASG12D that reveals a remarkable conformational change. The Switch I loop of one His-KRASG12D structure extends into the Switch I/II pocket of another His-KRASG12D in an adjacent unit cell to create an elaborate interface that is reminiscent of high-affinity protein-protein complexes. We explore the contributions of amino acids at this interface using alanine-scanning studies with alchemical free energy perturbation calculations based on explicit-solvent molecular dynamics simulations. Several interface amino acids were found to be hot spots as they contributed more than 1.5 kcal/mol to the protein-protein interaction. Computational analysis of the complex revealed the presence of two large binding pockets that possess physicochemical features typically found in pockets considered druggable. Small-molecule binding to these pockets may stabilize this autoinhibited structure of KRAS if it exists in cells to provide a new strategy to inhibit RAS signaling.

Cataract-associated connexin 46 mutation alters its interaction with calmodulin and function of hemichannels.

Connexin channels help maintain eye lens homeostasis and transparency. The G143R missense substitution in connexin (Cx) 46 is associated with congenital Coppock cataracts; however, the underlying molecular mechanism is largely unknown. Here, we report that compared with WT Cx46, the G143R substitution abolishes hemichannel conductance in Xenopus oocytes and in HeLa cells. Moreover, this substitution is dominant-negative and inhibits conductance of WT Cx46. CD analysis indicated that the substitution greatly reduces the α-helical structure of the intracellular Cx46 loop domain. Protein pulldown assays and isothermal titration calorimetry revealed that this Cx46 domain directly interacts with calmodulin (CaM) in a Ca2+-dependent fashion, an observation confirmed by immunofluorescent co-localization of Cx46 with CaM. Interestingly, the G143R substitution enhanced the Cx46-CaM interaction and attenuated its abolishment by Ca2+ depletion. Moreover, Cx46 increased dye influx, and the G143R substitution augmented this effect. Inhibition of Ca2+-mediated CaM activation blocked hemichannel permeability. The membrane potential plays a crucial role in Cx46 membrane permeability. We found that the activity of hemichannels is detectable under rest and hyperpolarization conditions but is eliminated with depolarization. These results suggested that the G143R substitution impairs voltage-dependent electrical conductance and alters membrane permeability mediated by Cx46 hemichannels. The latter likely is caused by the substitution-induced structural changes of the intracellular loop domain associated with the increased interaction with CaM and reduced Ca2+ sensitivity. The data suggest that the G143R-induced enhancement of the CaM-Cx46 interaction results in altered hemichannel activities and might be related to cataract formation.

Allogeneic Mesenchymal Stem Cells for Treatment of AKI after Cardiac Surgery.

AKI after cardiac surgery remains strongly associated with mortality and lacks effective treatment or prevention. Preclinical studies suggest that cell-based interventions may influence functional recovery. We conducted a phase 2, randomized, double-blind, placebo-controlled trial in 27 centers across North America to determine the safety and efficacy of allogeneic human mesenchymal stem cells (MSCs) in reducing the time to recovery from AKI after cardiac surgery. We randomized 156 adult subjects undergoing cardiac surgery with evidence of early AKI to receive intra-aortic MSCs (AC607; n=67) or placebo (n=68). The primary outcome was the time to recovery of kidney function defined as return of postintervention creatinine level to baseline. The median time to recovery of kidney function was 15 days with AC607 and 12 days with placebo (25th, 75th percentile range, 10-29 versus 6-21, respectively; hazard ratio, 0.81; 95% confidence interval, 0.53 to 1.24; P=0.32). We did not detect a significant difference between groups in 30-day all-cause mortality (16.7% with AC607; 11.8% with placebo) or dialysis (10.6% with AC607; 7.4% with placebo). At follow-up, 12 patients who received AC607 and six patients who received placebo had died. Rates of other adverse events did not differ between groups. In these patients with AKI after cardiac surgery, administration of allogeneic MSCs did not decrease the time to recovery of kidney function. Our results contrast with those in preclinical studies and provide important information regarding the potential effects of MSCs in this setting.

ß1-Subunit of the calcium-sensitive potassium channel modulates the pulmonary vascular smooth muscle cell response to hypoxia.

Accessory subunits associated with the calcium-sensitive potassium channel (BKCa), a major determinant of vascular tone, confer functional and anatomical diversity. The ß1 subunit increases Ca2+ and voltagesensitivity of the BKCa channel and is expressed exclusively in smooth muscle cells. Evidence supporting the physiological significance of the ß1 subunit includes the observations that murine models with deletion of the ß1 subunit are hypertensive and that humans with a gain-of-function ß1 mutation are at a decreased risk of diastolic hypertension. However, whether the ß1 subunit of the BKCa channel contributes to the low tone that characterizes the normal pulmonary circulation or modulates the pulmonary vascular response to hypoxia remains unknown. To determine the role of the BKCa channel ß1 subunit in the regulation of pulmonary vascular tone and the response to acute and chronic hypoxia, mice with deletion of the Kcnmb1 gene that encodes for the ß1 subunit ( Kcnmb1-/-) were placed in chronic hypoxia (10% O2) for 21-24 days. In normoxia, right ventricular systolic pressure (RVSP) did not differ between Kcnmb1+/+ (controls) and Kcnmb1-/- mice. After exposure to either acute or chronic hypoxia, RVSP was higher in Kcnmb1-/- mice compared with Kcnmb1+/+ mice, without increased vascular remodeling. ß1 subunit expression was predominantly confined to pulmonary artery smooth muscle cells (PASMCs) from vessels ≤ 150 µm. Peripheral PASMCs contracted collagen gels irrespective of ß1 expression. Focal adhesion expression and Rho kinase activity were greater in Kcnmb1-/- compared with Kcnmb1+/+ PASMCs. Compromised PASMC ß1 function may contribute to the heightened microvascular vasoconstriction that characterizes pulmonary hypertension.

A computational model for how the fast afterhyperpolarization paradoxically increases gain in regularly firing neurons.

The gain of a neuron, the number and frequency of action potentials triggered in response to a given amount of depolarizing injection, is an important behavior underlying a neuron's function. Variations in action potential waveform can influence neuronal discharges by the differential activation of voltage- and ion-gated channels long after the end of a spike. One component of the action potential waveform, the afterhyperpolarization (AHP), is generally considered an inhibitory mechanism for limiting firing rates. In dentate gyrus granule cells (DGCs) expressing fast-gated BK channels, large fast AHPs (fAHP) are paradoxically associated with increased gain. In this article, we describe a mechanism for this behavior using a computational model. Hyperpolarization provided by the fAHP enhances activation of a dendritic inward current (a T-type Ca2+ channel is suggested) that, in turn, boosts rebound depolarization at the soma. The model suggests that the fAHP may both reduce Ca2+ channel inactivation and, counterintuitively, enhance its activation. The magnitude of the rebound depolarization, in turn, determines the activation of a subsequent, slower inward current (a persistent Na+ current is suggested) limiting the interspike interval. Simulations also show that the effect of AHP on gain is also effective for physiologically relevant stimulation; varying AHP amplitude affects interspike interval across a range of "noisy" stimulus frequency and amplitudes. The mechanism proposed suggests that small fAHPs in DGCs may contribute to their limited excitability. NEW & NOTEWORTHY The afterhyperpolarization (AHP) is canonically viewed as a major factor underlying the refractory period, serving to limit neuronal firing rate. We recently reported that enhancing the amplitude of the fast AHP (fAHP) in a relatively slowly firing neuron (vs. fast spiking neurons) expressing fast-gated BK channels augments neuronal excitability. In this computational study, we present a novel, quantitative hypothesis for how varying the amplitude of the fAHP can, paradoxically, influence a subsequent spike tens of milliseconds later.

Clinicopathological case: progressive cognitive decline with gait disturbance in a steel worker.

A 57-year-old male steel plant worker presented with fatigue and altered liver function tests. Over the next two years, he developed cognitive decline, parkinsonism and seizures. This paper reports the clinicopathological conference at the 37th Edinburgh Advanced Neurology Course 2015 and outlines what we can learn from this case.

Knockout of the BK ß4-subunit promotes a functional coupling of BK channels and ryanodine receptors that mediate a fAHP-induced increase in excitability.

BK channels are large-conductance calcium- and voltage-activated potassium channels with diverse properties. Knockout of the accessory BK ß4-subunit in hippocampus dentate gyrus granule neurons causes BK channels to change properties from slow-gated type II channels to fast-gated type I channels that sharpen the action potential, increase the fast afterhyperpolarization (fAHP) amplitude, and increase spike frequency. Here we studied the calcium channels that contribute to fast-gated BK channel activation and increased excitability of ß4 knockout neurons. By using pharmacological blockers during current-clamp recording, we find that BK channel activation during the fAHP is dependent on ryanodine receptor activation. In contrast, L-type calcium channel blocker (nifedipine) affects the BK channel-dependent repolarization phase of the action potential but has no effect on the fAHP. Reducing BK channel activation during the repolarization phase with nifedipine, or during the fAHP with ryanodine, indicated that it is the BK-mediated increase of the fAHP that confers proexcitatory effects. The proexcitatory role of the fAHP was corroborated using dynamic current clamp. Increase or decrease of the fAHP amplitude during spiking revealed an inverse relationship between fAHP amplitude and interspike interval. Finally, we show that the seizure-prone ryanodine receptor gain-of-function (R2474S) knockin mice have an unaltered repolarization phase but larger fAHP and increased AP frequency compared with their control littermates. In summary, these results indicate that an important role of the ß4-subunit is to reduce ryanodine receptor-BK channel functional coupling during the fAHP component of the action potential, thereby decreasing excitability of dentate gyrus neurons.

Acute Treatment with the M-Channel (Kv7, KCNQ) Opener Retigabine Reduces the Long-Term Effects of Repetitive Blast Traumatic Brain Injuries.

We investigated whether pharmacological increase of "M-type" (KCNQ, Kv7) K + channel currents by the M-channel opener, retigabine (RTG), acutely after repetitive traumatic brain injuries (rTBIs), prevents or reduces their long-term detrimental effects. rTBIs were studied using a blast shock air wave mouse model. Animals were monitored by video and electroencephalogram (EEG) records for nine months after the last injury to assess the occurrence of post-traumatic seizures (PTS), post-traumatic epilepsy (PTE), sleep-wake cycle architecture alterations, and the power of the EEG signals. We evaluated the development of long-term changes in the brain associated with various neurodegenerative diseases in mice by examining transactive response DNA-binding protein 43 (TDP-43) expression and nerve fiber damage ~ 2 years after the rTBIs. We observed acute RTG treatment to reduce the duration of PTS and impair the development of PTE. Acute RTG treatment also prevented post-injury hypersomnia, nerve fiber damage, and cortical TDP-43 accumulation and translocation from the nucleus to the cytoplasm. Mice that developed PTE displayed impaired rapid eye movement (REM) sleep, and there were significant correlations between seizure duration and time spent in the different stages of the sleep-wake cycle. We observed acute RTG treatment to impair injury-induced reduction of age-related increase in gamma frequency power of the EGG, which has been suggested to be necessary for a healthy aged brain. The data show that RTG, administered acutely post-TBI, is a promising, novel therapeutic option to blunt/prevent several long-term effects of rTBIs. Furthermore, our results show a direct relationship between sleep architecture and PTE.

Molecular and functional aspects of menstruation in the macaque.

Much of our understanding of the molecular control of menstruation arises from laboratory models that experimentally recapitulate some, but not all, aspects of uterine bleeding observed in women. These models include: in vitro culture of endometrial explants or isolated endometrial cells, transplantation of human endometrial tissue into immunodeficient mice and the induction of endometrial breakdown in appropriately pretreated mice. Each of these models has contributed to our understanding of molecular and cellular mechanisms of menstruation, but nonhuman primates, especially macaques, are the animal model of choice for evaluating therapies for menstrual disorders. In this chapter we review some basic aspects of menstruation, with special emphasis on the macaque model and its relevance to the clinical issues of irregular and heavy menstrual bleeding (HMB).

Early histological changes in the porcine aortic media after thoracic stent-graft implantation.

PURPOSE: To describe the histological findings in the aortic wall 5 days after thoracic endovascular aortic repair (TEVAR) in a porcine model. METHODS: Two overlapping stent-grafts were implanted in each of 6 juvenile pigs, covering the entire descending thoracic aorta (DTA). On the 5(th) postoperative day, tissue samples were taken from the DTA in each animal. Medial thickness and medial necrosis were quantified and compared to measurements from the aortas of 6 control animals. RESULTS: Significant medial thinning was observed in stent-covered regions in the test animals. At the proximal landing zone, aortic wall thickness changed from 1387±68 to 782±74 µm within the covered aortic segment (p = 0.028); at the distal landing site, the wall thickness was 365±67 µm within the stent and 501±57 µm distally (p = 0.028). In the overlap zone, the aortic wall measured 524±122 vs. 1053±77 µm in native controls (p = 0.004). Aortic thickness proximal to the graft did not differ from the proximal region of native aortas (1468±96 vs. 1513±80 µm, p = 0.423), but the aorta was significantly thinner distal to the stent (707±38 vs. 815±52 µm, p = 0.004). Laminar necrosis constituted 38%±7% of the media in the proximal landing zone, 54%±4% in the overlap zone, and 46%±13% in the distal landing zone. CONCLUSION: In this porcine model, significant medial thinning and necrosis of the stented aorta was observed. The findings suggest an early phase of vulnerability of the aortic wall, before scarring and adaptive changes have strengthened the residual aorta.

Effects of Sublethal Organophosphate Toxicity and Anti-cholinergics on Electroencephalogram and Respiratory Mechanics in Mice.

Organophosphates are used in agriculture as insecticides but are potentially toxic to humans when exposed at high concentrations. The mechanism of toxicity is through antagonism of acetylcholinesterase, which secondarily causes excess activation of cholinergic receptors leading to seizures, tremors, respiratory depression, and other physiological consequences. Here we investigated two of the major pathophysiological effects, seizures and respiratory depression, using subcutaneous injection into mice of the organophosphate diisopropylfluorophosphate (DFP) at sublethal concentrations (2.1 mg/Kg) alone and co-injected with current therapeutics atropine (50 mg/Kg) or acetylcholinesterase reactivator HI6 (3 mg/Kg). We also tested a non-specific cholinergic antagonist dequalinium chloride (2 mg/Kg) as a novel treatment for organophosphate toxicity. Electroencephalogram (EEG) recordings revealed that DFP causes focal delta frequency (average 1.4 Hz) tonic spikes in the parietal region that occur transiently (lasting an average of 171 ± 33 min) and a more sustained generalized theta frequency depression in both parietal and frontal electrode that did not recover the following 24 h. DFP also caused behavioral tremors that partially recovered the following 24 h. Using whole body plethysmography, DFP revealed acute respiratory depression, including reduced breathing rates and tidal volumes, that partially recover the following day. Among therapeutic treatments, dequalinium chloride had the most potent effect on all physiological parameters by reducing acute EEG abnormalities and promoting a full recovery after 24 h from tremors and respiratory depression. Atropine and HI6 had distinct effects on EEGs. Co-treatment with atropine converted the acute 1.4 Hz tonic spikes to 3 Hz tonic spikes in the parietal electrode and promoted a partial recovery after 24 h from theta frequency and respiratory depression. HI6 fully removed the parietal delta spike increase and promoted a full recovery in theta frequency and respiratory depression. In summary, while all anticholinergic treatments promoted survival and moderated symptoms of DFP toxicity, the non-selective anti-cholinergic dequalinium chloride had the most potent therapeutic effects in reducing EEG abnormalities, moderating tremors and reducing respiratory depression.

Dequalinium chloride is an antagonists of α7 nicotinic acetylcholine receptors.

Dequalinium chloride has been used primarily as antiseptic compounds, but recently has been investigated for its effects on specific targets, including muscarinic acetylcholine receptors. Here we investigated dequalinium chloride as an antagonist to α7 nicotinic acetylcholine receptors. The pharmacological properties of dequalinium were established using cell lines stably co-transfected with the calcium-permeable human α7 nicotinic acetylcholine receptors and its chaperone NACHO, calcium dye fluorescent measurements or a calcium-sensitive protein reporter, and patch clamp recording of ionic currents. Using calcium dye fluorescence plate reader measurements, we find dequalinium chloride is an antagonist of α7 nicotinic acetylcholine receptors with an IC50 of 672 nM in response to activation with 500 µM acetylcholine chloride and positive allosteric modulator PNU-120596. However, using a membrane-tethered GCAMP7s calcium reporter allowed detection of α7-mediated calcium flux in the absence of PNU-120596. Using this approach revealed an IC50 of 157 nM for dequalinium on 300 µM acetylcholine-evoked currents. Using patch clamp recordings with 300 µM acetylcholine chloride and 10 µM PNU-120596, we find lower concentrations are sufficient to block ionic currents, with IC50 of 120 nM for dequalinium chloride and 54 nM for the related UCL 1684 compound. In summary, we find that dequalinium chloride and UCL1684, which are generally used to block SK-type potassium channels, are also highly effective antagonists of α7 nicotinic acetylcholine receptors. This finding, in combination with previous studies of muscarinic acetylcholine receptors, clearly establishes dequalinium compounds within the class of general anti-cholinergic antagonists.

BK channel ß1 subunits regulate airway contraction secondary to M2 muscarinic acetylcholine receptor mediated depolarization.

The large conductance calcium- and voltage-activated potassium channel (BK channel) and its smooth muscle-specific ß1 subunit regulate excitation­contraction coupling in many types of smooth muscle cells. However, the relative contribution of BK channels to control of M2- or M3-muscarinic acetylcholine receptor mediated airway smooth muscle contraction is poorly understood. Previously, we showed that knockout of the BK channel ß1 subunit enhances cholinergic-evoked trachea contractions. Here, we demonstrate that the enhanced contraction of the BK ß1 knockout can be ascribed to a defect in BK channel opposition of M2 receptor-mediated contractions. Indeed, the enhanced contraction of ß1 knockout is eliminated by specific M2 receptor antagonism. The role of BK ß1 to oppose M2 signalling is evidenced by a greater than fourfold increase in the contribution of L-type voltage-dependent calcium channels to contraction that otherwise does not occur with M2 antagonist or with ß1 containing BK channels. The mechanism through which BK channels oppose M2-mediated recruitment of calcium channels is through a negative shift in resting voltage that offsets, rather than directly opposes, M2-mediated depolarization. The negative shift in resting voltage is reduced to similar extents by BK ß1 knockout or by paxilline block of BK channels. Normalization of ß1 knockout baseline voltage with low external potassium eliminated the enhanced M2-receptor mediated contraction. In summary, these findings indicate that an important function of BK/ß1 channels is to oppose cholinergic M2 receptor-mediated depolarization and activation of calcium channels by restricting excitation­contraction coupling to more negative voltage ranges.

Modulation by the BK accessory ß4 subunit of phosphorylation-dependent changes in excitability of dentate gyrus granule neurons.

Large-conductance voltage- and calcium-activated potassium (BK) channels are large-conductance calcium- and voltage-activated potassium channels critical for neuronal excitability. Some neurons express so called fast-gated, type I BK channels. Other neurons express BK channels assembled with the accessory ß4 subunit conferring slow gating of type II BK channels. However, it is not clear how protein phosphorylation modulates these two distinct BK channel types. Using ß4-knockout mice, we compared fast- or slow-gated BK channels in response to changes in phosphorylation status of hippocampus dentate gyrus granule neurons. We utilized the selective PP2A/PP4 phosphatase inhibitor Fostriecin to study changes in action potential shape and firing properties of the neurons. In ß4-knockout neurons, Fostriecin increases BK current, speeds up BK channel activation and reduces action potential amplitudes. Fostriecin increases spiking during early components of an action potential train. In contrast, inhibition of BK channels through ß4 in wild-type neurons or by the BK channel inhibitor Paxilline opposes Fostriecin effects. Voltage clamp recordings of neurons reveal that Fostriecin increases both calcium and BK currents. However, Fostriecin does not activate BK α channels in transfected HEK293 cells lacking calcium channels. In summary, these results suggest that fast-gating, type I BK channels lacking ß4 can increase neuronal excitability in response to reduced phosphatase activity and activation of calcium channels. By opposing BK channel activation, the ß4 subunit plays an important role in moderating firing frequency regardless of changes in phosphorylation status.

Pollen Feeding Reduces Predation of Northern Corn Rootworm Eggs (Coleoptera: Chrysomelidae, Diabrotica barberi) by a Soil-Dwelling Mite (Acari: Laelapidae: Stratiolaelaps scimitus).

Landscape diversification with flowering plants can benefit pollinators and natural enemies, although insect pests can also use floral resources for nutrition and chemoprotection. Corn rootworms (Coleoptera: Chrysomelidae, Diabrotica spp.) are major pests of corn (Zea mays L.), and while subterranean larvae primarily feed on corn roots, adult rootworms commonly consume floral resources from other plant species. We quantified the species, density, and sex of adult corn Diabroticite rootworm beetles on wild and cultivated sunflower, corn, and squash, quantified pollen within the bodies of adult northern corn rootworms [NCR, D. barberi (Smith & Lawrence)], and investigated how consumption of sunflower and corn pollen by NCR adults impacted predation of their eggs by two soil-dwelling mites with different feeding specialization. NCR were the most common Diabroticite species on sunflower inflorescences and western corn rootworm (WCR, D. v. virgifera LeConte) were more abundant in corn and squash blossoms. Pollen feeding by NCR adults did not impact egg predation by omnivorous Tyrophagus putrescentiae (Schrank) (Acari: Sarcoptiformes, Acaridae), but predatory Stratiolaelaps scimitus (Womersley) (Acari: Mesostigmata, Laelapidae) ate eggs less frequently and took longer to feed on eggs from NCR females that had fed on sunflower pollen. This research suggests pollen feeding by adult NCR can impact predation of their eggs. While increasing plant diversity can benefit natural enemies and pest control within agroecosystems, it is important to consider how floral resources alter dietary preferences of biocontrol agents.

Neuroprotective Roles of the Adenosine A3 Receptor Agonist AST-004 in Mouse Model of Traumatic Brain Injury.

Traumatic brain injury (TBI) remains one of the greatest public health concerns with increasing morbidity and mortality rates worldwide. Our group reported that stimulation of astrocyte mitochondrial metabolism by P2Y1 receptor agonists significantly reduced cerebral edema and reactive gliosis in a TBI model. Subsequent data on the pharmacokinetics (PK) and rapid metabolism of these compounds suggested that neuroprotection was likely mediated by a metabolite, AST-004, which binding data indicated was an adenosine A3 receptor (A3R) agonist. The neuroprotective efficacy of AST-004 was tested in a control closed cortical injury (CCCI) model of TBI in mice. Twenty-four (24) hours post-injury, mice subjected to CCCI and treated with AST-004 (0.22 mg/kg, injected 30 min post-trauma) exhibited significantly less secondary brain injury. These effects were quantified with less cell death (PSVue794 fluorescence) and loss of blood brain barrier breakdown (Evans blue extravasation assay), compared to vehicle-treated TBI mice. TBI-treated mice also exhibited significantly reduced neuroinflammatory markers, glial-fibrillary acidic protein (GFAP, astrogliosis) and ionized Ca2+-binding adaptor molecule 1 (Iba1, microgliosis), both at the mRNA (qRT-PCR) and protein (Western blot and immunofluorescence) levels, respectively. Four (4) weeks post-injury, both male and female TBI mice presented a significant reduction in freezing behavior during contextual fear conditioning (after foot shock). AST-004 treatment prevented this TBI-induced impairment in male mice, but did not significantly affect impairment in female mice. Impairment of spatial memory, assessed 24 and 48 h after the initial fear conditioning, was also reduced in AST-004-treated TBI-male mice. Female TBI mice did not exhibit memory impairment 24 and 48 h after contextual fear conditioning and similarly, AST-004-treated female TBI mice were comparable to sham mice. Finally, AST-004 treatments were found to increase in vivo ATP production in astrocytes (GFAP-targeted luciferase activity), consistent with the proposed mechanism of action. These data reveal AST-004 as a novel A3R agonist that increases astrocyte energy production and enhances their neuroprotective efficacy after brain injury.

An african-specific functional polymorphism in KCNMB1 shows sex-specific association with asthma severity.

A highly heritable and reproducible measure of asthma severity is baseline pulmonary function. Pulmonary function is largely determined by airway smooth muscle (ASM) tone and contractility. The large conductance, voltage and calcium-activated potassium (BK) channel negatively regulates smooth muscle tone and contraction in ASM. The modulatory subunit of BK channels, the beta1-subunit, is critical for proper activation of BK channels in smooth muscle and has shown sex hormone specific regulation. We hypothesized that KCNMB1 genetic variants in African Americans may underlie differences in bronchial smooth muscle tone and thus pulmonary function, possibly in a sex-specific manner. Through resequencing of the KCNMB1 gene we identified several common variants including a novel African-specific coding polymorphism (C818T, R140W). The C818T SNP and four other KCNMB1 variants were genotyped in two independent groups of African American asthmatics (n = 509) and tested for association with the pulmonary function measure--forced expiratory volume (FEV(1)) % of predicted value. The 818T allele is associated with a clinically significant decline (-13%) in FEV(1) in both cohorts of asthmatics among males but not females (P(combined) = 0.0003). Patch clamp electrophysiology studies of the BK channel expressed with the 140Trp variant of the beta1-subunit demonstrated significantly reduced channel openings, predicted by the loss of pulmonary function observed. African American male asthmatics carrying the 818T allele (10% of population) are potentially at risk for greater airway obstruction and increased asthma morbidity. Female asthmatics may be insulated from the deleterious effects of the 818T allele by estrogen-mediated upregulation in BK channel activity.

Coordination of multidisciplinary resources for vaccination of egg-allergic individuals during an H1N1 (novel) influenza pandemic.

Egg allergy is a contraindication for influenza vaccination because of the presence of trace egg protein. Although recent reports suggest that egg-allergic individuals can be safely immunized with seasonal influenza vaccine, the safety of H1N1 influenza vaccine in this context has not previously been reported. This study was designed to investigate how to safely immunize high-risk populations during the H1N1 influenza pandemic for whom the vaccine would be contraindicated. Vaccination of egg-allergic children was conducted at a large, multispecialty practice. Pediatricians raised awareness of vaccination and referred egg-allergic individuals to allergists. Allergists performed skin testing to H1N1 vaccine and, if negative, performed graded vaccine desensitizations in the office. Mass immunization of egg-allergic individuals was achieved within a 3-week period after receipt of vaccine. Sixty-three children (average age, 3.7 years) were evaluated and skin tested to the H1N1 influenza vaccine. All had a clinical history of egg allergy and recent positive skin-prick to egg. All had negative skin-prick test to the H1N1 vaccine and received graded challenge with three divided doses. All 62 children were successfully immunized without allergic reactions. Vaccination of egg-allergic individuals during a pandemic can be safely and efficiently accomplished with the coordination of multidisciplinary resources. Although this was conducted at a multispecialty practice, we believe that this invaluable public health service can be done outside of this context. Allergists, with the help of local pediatricians and urgent care centers or hospitals, can effectively mobilize to help protect the most vulnerable populations during pandemics.

Influence of a Neonicotinoid Seed Treatment on a Nontarget Herbivore of Soybean (Twospotted Spider Mite) and Diet Switching by a Co-occurring Omnivore (Western Flower Thrips).

Insecticidal neonicotinoid seed treatments are a common agricultural insect pest management strategy; however, effects on nontarget pests and omnivorous arthropods are understudied. We used a series of experiments to evaluate impacts of the neonicotinoid seed treatment thiamethoxam on densities of herbivorous twospotted spider mites (Tetranychus urticae Koch [Acari: Tetranychidae]) and feeding behavior of western flower thrips (Frankliniella occidentalis Pergande [Thysanoptera: Thripidae]), an omnivore that feeds on spider mite eggs but is also a significant plant pest. Spider mite densities were higher on neonicotinoid-treated soybeans, but only when mites were not spatially confined. We then examined how availability of thiamethoxam-treated food items (i.e., eggs from spider mites reared on treated soybeans, soybean leaf discs, or a combination of the two), and previous exposure to thiamethoxam-treated soybean impacted thrips feeding. Regardless of the presence of leaf tissue, thrips consumed fewer spider mite eggs laid by females reared on treated soybeans, suggesting spider mite eggs can serve as poisoned prey. Overall, thrips consumed less treated soybean leaf tissue, and thrips on treated leaf discs had a lower percentage of herbivorous feeding events and consumed more nontreated spider mite eggs, indicating a dietary shift from herbivory to predation. The neonicotinoid status of spider mite eggs and prior exposure of thrips also caused shifts in the number and size of leaf scars, likely as a result of altered foraging behavior and/or movement. Shifts between herbivory and predation have implications for thrips damage, virus transmission, and pest management, especially in systems with mixtures of nontreated and neonicotinoid-treated plants.