Sorghum 3-Deoxyanthocyanidin Flavonoids Confer Resistance against Corn Leaf Aphid.

In this study we examined the role of sorghum flavonoids in providing resistance against corn leaf aphid (CLA) Rhopalosiphum maidis. In sorghum, accumulation of these flavonoids is regulated by a MYB transcription factor, yellow seed1 (y1). Functional y1 alleles accumulate 3-deoxyflavonoids (3-DFs) and 3-deoxyanthocyanidins (3-DAs) whereas null y1 alleles fail to accumulate these compounds. We found that significantly higher numbers of alate CLA adults colonized null y1 plants as compared to functional y1 plants. Controlled cage experiments and pairwise choice assays demonstrated that apterous aphids preferred to feed and reproduce on null y1 plants. These near-isogenic sorghum lines do not differ in their epicuticular wax content and were also devoid of any leaf trichomes. Significantly higher mortality of CLA was observed on artificial aphid diet supplemented with flavonoids obtained from functional y1 plants as compared to null y1 plants or the relevant controls. Our results demonstrate that the proximate mechanism underlying the deleterious effects on aphids is y1-regulated flavonoids which are important defense compounds against CLA.

miRNA-mediated auxin signalling repression during Vat-mediated aphid resistance in Cucumis melo.

Resistance to Aphis gossypii in melon is attributed to the presence of the single dominant R gene virus aphid transmission (Vat), which is biologically expressed as antibiosis, antixenosis and tolerance. However, the mechanism of resistance is poorly understood at the molecular level. Aphid-induced transcriptional changes, including differentially expressed miRNA profiles that correspond to resistance interaction have been reported in melon. The potential regulatory roles of miRNAs in Vat-mediated aphid resistance were further revealed by identifying the specific miRNA degradation targets. A total of 70 miRNA:target pairs, including 28 novel miRNA:target pairs, for the differentially expressed miRNAs were identified: 11 were associated with phytohormone regulation, including six miRNAs that potentially regulate auxin interactions. A model for a redundant regulatory system of miRNA-mediated auxin insensitivity is proposed that incorporates auxin perception, auxin modification and auxin-regulated transcription. Chemically inhibiting the transport inhibitor response-1 (TIR-1) auxin receptor in susceptible melon tissues provides in vivo support for the model of auxin-mediated impacts on A. gossypii resistance.

Phloem ultrastructure and pressure flow: Sieve-Element-Occlusion-Related agglomerations do not affect translocation.

Since the first ultrastructural investigations of sieve tubes in the early 1960s, their structure has been a matter of debate. Because sieve tube structure defines frictional interactions in the tube system, the presence of P protein obstructions shown in many transmission electron micrographs led to a discussion about the mode of phloem transport. At present, it is generally agreed that P protein agglomerations are preparation artifacts due to injury, the lumen of sieve tubes is free of obstructions, and phloem flow is driven by an osmotically generated pressure differential according to Münch's classical hypothesis. Here, we show that the phloem contains a distinctive network of protein filaments. Stable transgenic lines expressing Arabidopsis thaliana Sieve-Element-Occlusion-Related1 (SEOR1)-yellow fluorescent protein fusions show that At SEOR1 meshworks at the margins and clots in the lumen are a general feature of living sieve tubes. Live imaging of phloem flow and flow velocity measurements in individual tubes indicate that At SEOR1 agglomerations do not markedly affect or alter flow. A transmission electron microscopy preparation protocol has been generated showing sieve tube ultrastructure of unprecedented quality. A reconstruction of sieve tube ultrastructure served as basis for tube resistance calculations. The impact of agglomerations on phloem flow is discussed.

The broccoli (Brassica oleracea) phloem tissue proteome.

BACKGROUND: The transport of sugars, hormones, amino acids, proteins, sugar alcohols, and other organic compounds from the sites of synthesis to the sites of use or storage occurs through the conducting cells of the phloem. To better understand these processes a comprehensive understanding of the proteins involved is required. While a considerable amount of data has been obtained from proteomic analyses of phloem sap, this has mainly served to identify the soluble proteins that are translocated through the phloem network. RESULTS: In order to obtain more comprehensive proteomic data from phloem tissue we developed a simple dissection procedure to isolate phloem tissue from Brassica oleracea. The presence of a high density of phloem sieve elements was confirmed using light microscopy and fluorescently labeled sieve element-specific antibodies. To increase the depth of the proteomic analysis for membrane bound and associated proteins, soluble proteins were extracted first and subsequent extractions were carried out using two different detergents (SDS and CHAPSO). Across all three extractions almost four hundred proteins were identified and each extraction method added to the analysis demonstrating the utility of an approach combining several extraction protocols. CONCLUSIONS: The phloem was found to be enriched in proteins associated with biotic and abiotic stress responses and structural proteins. Subsequent expression analysis identified a number of genes that appear to be expressed exclusively or at very high levels in phloem tissue, including genes that are known to express specifically in the phloem as well as novel phloem genes.

Cucumis melo microRNA expression profile during aphid herbivory in a resistant and susceptible interaction.

Aphis gossypii resistance in melon (Cucumis melo) is due to the presence of a single dominant virus aphid transmission (Vat) gene belonging to the nucleotide-binding site leucine-rich repeat family of resistance genes. Significant transcriptional reprogramming occurs in Vat(+) plants during aphid infestation as metabolism shifts to respond to this biotic stress. MicroRNAs (miRNAs) are involved in the regulation of many biotic stress responses. The role of miRNAs was investigated in response to aphid herbivory during both resistant and susceptible interactions. Small RNA (smRNA) libraries were constructed from bulked leaf tissues of a Vat(+) melon line following early and late aphid infestations. Sequence analysis indicated that the expression profiles of conserved and newly identified miRNAs were altered during different stages of aphid herbivory. These results were verified by quantitative polymerase chain reaction experiments in both resistant Vat(+) and susceptible Vat(-) interactions. The comparative analyses revealed that most of the conserved miRNA families were differentially regulated during the early stages of aphid infestation in the resistant and susceptible interactions. Along with the conserved miRNA families, 18 cucurbit-specific miRNAs were expressed during the different stages of aphid herbivory. The comparison of the miRNA profiles in the resistant and susceptible interactions provides insight into the miRNA-dependent post-transcriptional gene regulation in Vat-mediated resistance.

Arabidopsis P-protein filament formation requires both AtSEOR1 and AtSEOR2.

The structure-function relationship of proteinaceous filaments in sieve elements has long been a source of investigation in order to understand their role in the biology of the phloem. Two phloem filament proteins AtSEOR1 (At3g01680.1) and AtSEOR2 (At3g01670.1) in Arabidopsis have been identified that are required for filament formation. Immunolocalization experiments using a phloem filament-specific monoclonal antibody in the respective T-DNA insertion mutants provided an initial indication that both proteins are necessary to form phloem filaments. To investigate the relationship between these two proteins further, green fluorescent protein (GFP)-AtSEO fusion proteins were expressed in Columbia wild-type and T-DNA insertion mutants. Analysis of these mutants by confocal microscopy confirmed that phloem filaments could only be detected in the presence of both proteins, indicating that despite significant sequence homology the proteins are not functionally redundant. Individual phloem filament protein subunits of AtSEOR1 and AtSEOR2 were capable of forming homodimers, but not heterodimers in a yeast two-hybrid system. The absence of phloem filaments in phloem sieve elements did not result in gross alterations of plant phenotype or affect basal resistance to green peach aphid (Myzus persicae).

Nicotinamide Inhibits Aphid Fecundity and Impacts Survival.

Nicotinamide (NAM) alters behavior in C. elegans and Drosophila, serving as an agonist of TRPV channels affecting sensory neurons and mimicking the mode of action of insecticides used to control phloem-feeding insects. The impact of NAM on green peach aphid (Myzus persicae) behaviors was assessed in artificial diet assays and foliar applications to Arabidopsis plants. Aphids feeding on artificial diets supplemented with NAM impaired stylet movement causing feeding interruptions and ultimately starvation and death. Aphid feeding behaviors were negatively impacted on NAM sprayed plants at concentrations as low as 2.5 mM leading to increased mortality. In choice assays with NAM sprayed leaves aphids showed clear preference for untreated control leaves. NAM is an intermediate in the NAD salvage pathway that should accumulate in nicotinamidase (nic) mutants. LC-MS analysis showed NAM accumulates 60-fold in nic-1-1 Arabidopsis mutants as compared with Col-0. Aphid reproductive potential was significantly decreased on nic-1-1 mutant plants, resulting in a smaller colony size and arrested population development. The results support the hypothesis that dietary NAM causes behavioral changes in aphids, including altered feeding, reduced reproduction, and increased mortality. NAM is thought to bind to TRPV channels causing overstimulation of sensory neurons in the aphid feeding apparatus.

Single-Dose Pharmacokinetic Study of Diphenhydramine HCl in Children and Adolescents.

Diphenhydramine pharmacokinetics were characterized following a single oral dose in children aged 2 to 17 years using a weight- and age-based dosing schedule with more tiers than the current age-based dosing schedule recommended by the nonprescription drug monograph. This study was conducted in 42 subjects, aged 2 to 17 years. Doses were based on a weight-age dosing schedule, ranging from 6.25 to 50 mg. An oral dose was administered with water about 2 hours after a light breakfast. Plasma samples were obtained up to 48 hours after dosing and analyzed for diphenhydramine. Pharmacokinetic parameters were estimated using noncompartmental methods, and the relationship of oral clearance with age was assessed using linear regression. Over an 8-fold range of doses, Cmax and AUC increased ∼90 % to ∼140% across age groups, with a similar Tmax (1.5 hours). Oral CL/F increased with age, but after allometric scaling, no maturation-related change in CL/F was apparent. Mild somnolence was the most commonly reported adverse event (95% of the subjects). A weight-age dosing schedule using an 8-fold range of doses achieved Cmax and AUC that increased about 2-fold across age groups. No effect of maturation on CL/F was observed after allometric scaling.

Brompheniramine and Chlorpheniramine Pharmacokinetics Following Single-Dose Oral Administration in Children Aged 2 to 17 Years.

Two pediatric studies characterized brompheniramine and chlorpheniramine pharmacokinetics in a total of 72 subjects, aged 2 to 17 years. A single age-/weight-based oral dose, ranging from 1 to 4 mg, was administered with 2 to 6 oz of water at least 2 hours after a light breakfast. Plasma samples were obtained before and for 72 hours after dosing and analyzed using high-pressure liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters were estimated using noncompartmental methods; relationships with age were assessed using linear regression. Results indicated that for brompheniramine and chlorpheniramine, Cmax was similar across age groups, although it tended to occur earlier in the youngest group. AUC was ∼15% to 30% higher in the oldest age group. As expected, CLo and Vz /F increased with age; however, following allometric scaling, no age-related differences existed. Because the increase with age for both parameters was similar, no age-related differences in t1/2,z existed (∼15 hours). Overall, the single doses were well tolerated. Sedation was the most common reported AE and appeared to be more prevalent in the 2- to 5-year-old group. Overall, these results indicate that an age/weight dosing nomogram using a 4-fold range of doses achieves similar Cmax and AUC.

Simultaneous determination of dextromethorphan, dextrorphan, and guaifenesin in human plasma using semi-automated liquid/liquid extraction and gradient liquid chromatography tandem mass spectrometry.

A method for the simultaneous determination of dextromethorphan (DEX), dextrorphan (DET), and guaifenesin (GG) in human plasma was developed, validated, and applied to determine plasma concentrations of these compounds in samples from six clinical pharmacokinetic (PK) studies. Semi-automated liquid handling systems were used to perform the majority of the sample manipulation including liquid/liquid extraction (LLE) of the analytes from human plasma. Stable-isotope-labeled analogues were utilized as internal standards (ISTDs) for each analyte to facilitate accurate and precise quantification. Extracts were analyzed using gradient liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Use of semi-automated LLE with LC-MS/MS proved to be a very rugged and reliable approach for analysis of more than 6200 clinical study samples. The lower limit of quantification was validated at 0.010, 0.010, and 1.0 ng/mL of plasma for DEX, DET, and GG, respectively. Accuracy and precision of quality control (QC) samples for all three analytes met FDA Guidance criteria of +/-15% for average QC accuracy with coefficients of variation less than 15%. Data from the thorough evaluation of the method during development, validation, and application are presented to characterize selectivity, linearity, over-range sample analysis, accuracy, precision, autosampler carry-over, ruggedness, extraction efficiency, ionization suppression, and stability. Pharmacokinetic data are also provided to illustrate improvements in systemic drug and metabolite concentration-time profiles that were achieved by formulation optimization.

Small RNA Regulators of Plant-Hemipteran Interactions: Micromanagers with Versatile Roles.

Non-coding small RNAs (sRNAs) in plants have important roles in regulating biological processes, including development, reproduction, and stress responses. Recent research indicates significant roles for sRNA-mediated gene silencing during plant-hemipteran interactions that involve all three of these biological processes. Plant responses to hemipteran feeding are determined by changes in the host transcriptome that appear to be fine-tuned by sRNAs. The role of sRNA in plant defense responses is complex. Different forms of sRNAs, with specific modes of action, regulate changes in the host transcriptome primarily through post-transcriptional gene silencing and occasionally through translational repression. Plant genetic resistance against hemipterans provides a model to explore the regulatory roles of sRNAs in plant defense. Aphid-induced sRNA expression in resistance genotypes delivers a new paradigm in understanding the regulation of R gene-mediated resistance in host plants. Unique sRNA profiles, including changes in sRNA biogenesis and expression can also provide insights into susceptibility to insect herbivores. Activation of phytohormone-mediated defense responses against insect herbivory is another hallmark of this interaction, and recent studies have shown that regulation of phytohormone signaling is under the control of sRNAs. Hemipterans feeding on resistant plants also show changes in insect sRNA profiles, possibly influencing insect development and reproduction. Changes in insect traits such as fecundity, host range, and resistance to insecticides are impacted by sRNAs and can directly contribute to the success of certain insect biotypes. In addition to causing direct damage to the host plant, hemipteran insects are often vectors of viral pathogens. Insect anti-viral RNAi machinery is activated to limit virus accumulation, suggesting a role in insect immunity. Virus-derived long sRNAs strongly resemble insect piRNAs, leading to the speculation that the piRNA pathway is induced in response to viral infection. Evidence for robust insect RNAi machinery in several hemipteran species is of immense interest and is being actively pursued as a possible tool for insect control. RNAi-induced gene silencing following uptake of exogenous dsRNA was successfully demonstrated in several hemipterans and the presence of sid-1 like genes support the concept of a systemic response in some species.

Guaifenesin Pharmacokinetics Following Single-Dose Oral Administration in Children Aged 2 to 17 Years.

This study characterized guaifenesin pharmacokinetics in children aged 2 to 17 years (n = 40) who received a single oral dose of guaifenesin (age-based doses of 100-400 mg) 2 hours after breakfast. Plasma samples were obtained before and for 8 hours after dosing and analyzed for guaifenesin using liquid chromatography-tandem mass spectrometry. Pharmacokinetic parameters were estimated using noncompartmental methods, relationships with age were assessed using linear regression, and dose proportionality was assessed on 95% confidence intervals. Based on the upper dose recommended in the monograph (for both children and adolescents), area under the curve from time zero to infinity and maximum plasma concentration both increased with age. However, when comparing the upper dose for children aged 2 to 11 years with the lower dose for adolescents aged 12 to 17 years, similar systemic exposure was observed. As expected due to increasing body size, oral clearance (CLo ) and terminal volume of distribution (Vz /F) increased with age. Due to a larger increase in Vz /F than CLo , an increase in terminal exponential half-life was also observed. Allometric scaling indicated no maturation-related changes in CLo and Vz /F.

Influence of coadministration on the pharmacokinetics of azimilide dihydrochloride and digoxin.

The influence of coadministration on digoxin and azimilide pharmacokinetics/pharmacodynamics was assessed in a randomized, 3-way crossover study in 18 healthy men. Serial blood and urine samples were obtained for azimilide and digoxin quantitation. Treatment effects on pharmacokinetics were assessed using analysis of variance. The relationship between azimilide blood concentrations and QT(c) prolongation was characterized by an E(max) model. Effects of coadministration on pharmacodynamics were assessed using a mechanistic-based inhibition model. Azimilide pharmacokinetics was unaffected by digoxin, except for a 36% increase in CL(r) (P = .0325), with no change in CL(o). Digoxin pharmacokinetics was unaffected by azimilide, except for a 21% increase in C(max) (P = .0176) and a 10% increase in AUC(tau) (P = .0121). Digoxin coadministration increased the apparent EC(50) with no effect on E(max), consistent with competitive inhibition (K(i) = 0.899 ng/mL). The pharmacokinetic and pharmacodynamic changes observed upon coadministration were small and are not expected to be clinically important.

Effects of Ketoconazole on the Pharmacokinetics of Lenvatinib (E7080) in Healthy Participants.

BACKGROUND: Lenvatinib is an oral, multitargeted, tyrosine kinase inhibitor under clinical investigation in solid tumors. In vitro evidence indicates that lenvatinib metabolism may be modulated by ketoconazole, an inhibitor of CYP3A4 and p-glycoprotein. METHODS: In this Phase I, single-center, randomized, open-label, two-period, crossover study, healthy adults (18-55 years; N = 18) were randomized to one of two sequences (ketoconazole → placebo or vice versa). Ketoconazole (400 mg) or placebo was administered orally once daily for 18 days; a 5 mg dose of lenvatinib was orally administered on Day 5 of each treatment period. Blood samples were collected over 14 days and lenvatinib plasma concentrations measured by high-performance liquid chromatography/tandem mass spectrometry. RESULTS: Systemic exposure to lenvatinib increased slightly (15-19%) with coadministration of ketoconazole. Although the 90% confidence interval (CI) for area under the plasma concentration-time curve (AUC) was within the prespecified bioequivalence interval of 80-125%, Cmax slightly exceeded the 125% CI bound (134%). No changes in tmax, tlag, or t½ were observed. Thirteen subjects (72%) experienced treatment-emergent adverse events (11 mild, 2 moderate), most commonly headache (22%) and diarrhea (17%). CONCLUSIONS: Lenvatinib exposure was slightly increased by ketoconazole; however, the magnitude of the change was relatively small, and likely not clinically meaningful.

Live imaging of companion cells and sieve elements in Arabidopsis leaves.

The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.

Influence of azimilide on CYP2C19-mediated metabolism.

The purpose of this study was to assess the influence of multiple-dose oral administration of azimilide dihydrochloride on CYP2C19-mediated metabolism. A two-period, randomized crossover study was conducted in 40 healthy male subjects who were phenotyped as extensive CYP2C19 metabolizers. Oral doses of placebo or 125 mg of azimilide dihydrochloride were administered every 12 hours for 3 days, followed by every 24 hours for 5 days; 20 mg omeprazole was coadministered on Day 8. Blood or plasma samples were obtained over 24 hours and analyzed for azimilide or omeprazole/5-hydroxyomeprazole using high-performance liquid chromatography with tandem mass spectrometry. Data were analyzed using "noncompartmental" analysis. Azimilide blood concentrations observed in this study were similar to those previously observed at steady state in patients. Based on AUC(m)/AUC(p) for omeprazole, azimilide does not significantly inhibit CYP2C19-mediated metabolism (90% confidence interval [CI] = 104%-111%). For 5-hydroxyomeprazole, no significant changes in pharmacokinetics were observed. For omeprazole, a statistically significant decrease ( approximately 12%) was observed for AUC. However, this change was small and is not expected to be clinically important since the CI was contained within those used to establish bioequivalence. These results indicate that azimilide does not inhibit CYP2C19-mediated metabolism. Since this isozyme had the lowest in vitro IC(50) values for the cytochrome P450s most commonly involved with the metabolism of drugs (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4), azimilide-related drug interactions mediated via these isozymes are not anticipated.

Effect of mild and moderate hepatic impairment on azimilide pharmacokinetics following single dose oral administration.

Azimilide dihydrochloride (75-125 mg/day) is currently being developed for use in prolonging the time to recurrence of atrial fibrillation/flutter and for reducing the frequency of shocks in patients with an implantable cardioverting defibrillator. This study investigated the influence of mild and moderate hepatic impairment on azimilide pharmacokinetics. Six subjects each with mild and moderate hepatic impairment (Child-Pugh grades A and B, respectively) were age, weight, smoking status, and gender-matched to a healthy subject (total N = 24). Each subject was administered a single, oral dose of 100 mg azimilide dihydrochloride following an overnight fast. Blood/plasma and urine samples were collected up to 28 days and over 9 days, respectively, and analyzed using HPLC with MS/MS or UV detection. For azimilide, most parameters in subjects with mild to moderate hepatic impairment were within 25% of those observed in matched healthy subjects, with no statistically significant differences observed. For F-1292 (major metabolite in plasma), a significant decrease in AUC was observed in subjects with moderate hepatic impairment, secondary to an increase in renal clearance (CL(r)). Based on these results, no a priori dosage adjustment is required in subjects with mild to moderate hepatic impairment.

Effect of rifampicin on the pharmacokinetics of lenvatinib in healthy adults.

BACKGROUND AND OBJECTIVES: Lenvatinib is an oral, multitargeted tyrosine kinase inhibitor under clinical investigation in solid tumours. This study evaluated the influence of P-glycoprotein (P-gp) inhibition (single-dose rifampicin) and simultaneous cytochrome P450 3A4 (CYP3A4)/P-gp induction (multiple-dose rifampicin) on lenvatinib pharmacokinetics. METHODS: This Phase I, single-centre, single-dose (lenvatinib mesylate 24 mg), open-label, sequential study enrolled 15 healthy volunteers. Three regimens were administered over three periods: Period (P) 1 (Days 1-8), P2 (Days 15-22) and P3 (Days 29-50), with a 14-day (first dose) and 28-day (second dose) washout period after lenvatinib mesylate administration (Day 1, Day 15 and Day 43). In P2, a single oral dose of rifampicin (600 mg) was coadministered with lenvatinib. In P3, rifampicin was administered daily (600 mg) for 21 days (Days 29-49). Serial blood samples were collected, and plasma concentrations of total (protein bound + unbound) and free (unbound) lenvatinib and total metabolites (M1, M2, M3 and M5) were measured by validated high-performance liquid chromatography/tandem mass spectrometry. RESULTS: Single-dose rifampicin (P-gp inhibition) increased area under the plasma concentration-time curve from time zero to infinity (AUC0-∞) of free and total lenvatinib by 32 and 31 %, respectively. Multiple-dose rifampicin (simultaneous P-gp and CYP3A4 induction) decreased lenvatinib AUC0-∞ (total: 18 %; free: 9 %). Treatment-emergent adverse events were mild or moderate and occurred in 7 subjects (47 %). CONCLUSION: Lenvatinib exposure was increased by P-gp inhibition; however, based on free concentrations, simultaneous P-gp and CYP3A4 induction results met the prespecified bioequivalence 90 % confidence interval. Overall, the magnitude of these changes was relatively small, and likely not clinically meaningful.

Doxylamine pharmacokinetics following single dose oral administration in children ages 2-17 years.

To characterize doxylamine pharmacokinetics in children. This study was conducted in 41 subjects, ages 2-17 years. Doxylamine succinate doses based on age/weight ranged from 3.125 to 12.5 mg. A single oral dose was administered with 2 to 4 oz. of water or decaffeinated beverages ∼2 hours after a light breakfast. Plasma samples were obtained before and for 72 hours after dosing and analyzed for doxylamine using HPLC MS/MS. Pharmacokinetic parameters were estimated using non-compartmental methods and relationships with age were assessed using linear regression. Over the fourfold dose range, Cmax was similar while AUC increased only 60%, although not statistically significant (P-value = 0.0517). As expected due to increasing body size, CLo and Vz /F increased with age. Due to a similar increase with age for Clo and Vz /F, no age-related differences in t1/2,z were observed (∼16 hours). Allometric scaling indicated no maturation related changes in CLo ; although Vz /F remained age-dependent, the predicted range decreased ∼70%. Overall, the single doses were well tolerated. Somnolence was the most common reported AE with no apparent differences in incidence noted with age. An age/weight dosing nomogram utilizing a fourfold range of doses achieves similar Cmax , whereas AUC increases only 60%.

Expression of small RNA in Aphis gossypii and its potential role in the resistance interaction with melon.

BACKGROUND: The regulatory role of small RNAs (sRNAs) in various biological processes is an active area of investigation; however, there has been limited information available on the role of sRNAs in plant-insect interactions. This study was designed to identify sRNAs in cotton-melon aphid (Aphis gossypii) during the Vat-mediated resistance interaction with melon (Cucumis melo). METHODOLOGY/PRINCIPAL FINDINGS: The role of miRNAs was investigated in response to aphid herbivory, during both resistant and susceptible interactions. sRNA libraries made from A. gossypii tissues feeding on Vat⁺ and Vat⁻ plants revealed an unexpected abundance of 27 nt long sRNA sequences in the aphids feeding on Vat⁺ plants. Eighty-one conserved microRNAs (miRNAs), twelve aphid-specific miRNAs, and nine novel candidate miRNAs were also identified. Plant miRNAs found in the aphid libraries were most likely ingested during phloem feeding. The presence of novel miRNAs was verified by qPCR experiments in both resistant Vat⁺ and susceptible Vat⁻ interactions. The comparative analyses revealed that novel miRNAs were differentially regulated during the resistant and susceptible interactions. Gene targets predicted for the miRNAs identified in this study by in silico analyses revealed their involvement in morphogenesis and anatomical structure determination, signal transduction pathways, cell differentiation and catabolic processes. CONCLUSION/SIGNIFICANCE: In this study, conserved and novel miRNAs were reported in A. gossypii. Deep sequencing data showed differences in the abundance of miRNAs and piRNA-like sequences in A. gossypii. Quantitative RT-PCR revealed that A. gossypii miRNAs were differentially regulated during resistant and susceptible interactions. Aphids can also ingest plant miRNAs during phloem feeding that are stable in the insect.