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1.  A Chrysodeixis chalcites Single-Nucleocapsid Nucleopolyhedrovirus Population from the Canary Islands Is Genotypically Structured To Maximize Survival 
Applied and Environmental Microbiology  2013;79(24):7709-7718.
A Chrysodeixis chalcites single-nucleocapsid nucleopolyhedrovirus wild-type isolate from the Canary Islands, Spain, named ChchSNPV-TF1 (ChchTF1-wt), appears to have great potential as the basis for a biological insecticide for control of the pest. An improved understanding of the genotypic structure of this wild-type strain population should facilitate the selection of genotypes for inclusion in a bioinsecticidal product. Eight genetically distinct genotypes were cloned in vitro: ChchTF1-A to ChchTF1-H. Quantitative real-time PCR (qPCR) analysis confirmed that ChchTF1-A accounted for 36% of the genotypes in the wild-type population. In bioassays, ChchTF1-wt occlusion bodies (OBs) were significantly more pathogenic than any of the component single-genotype OBs, indicating that genotype interactions were likely responsible for the pathogenicity phenotype of wild-type OBs. However, the wild-type population was slower killing and produced higher OB yields than any of the single genotypes alone. These results strongly suggested that the ChchTF1-wt population is structured to maximize its transmission efficiency. Experimental OB mixtures and cooccluded genotype mixtures containing the most abundant and the rarest genotypes, at frequencies similar to those at which they were isolated, revealed a mutualistic interaction that restored the pathogenicity of OBs. In OB and cooccluded mixtures containing only the most abundant genotypes, ChchTF1-ABC, OB pathogenicity was even greater than that of wild-type OBs. The ChchTF1-ABC cooccluded mixture killed larvae 33 h faster than the wild-type population and remained genotypically and biologically stable throughout five successive passages in vivo. In conclusion, the ChchTF1-ABC mixture shows great potential as the active ingredient of a bioinsecticide to control C. chalcites in the Canary Islands.
PMCID: PMC3837827  PMID: 24096419
2.  Mixture-Amount Design and Response Surface Modeling to Assess the Effects of Flavonoids and Phenolic Acids on Developmental Performance of Anastrepha ludens 
Journal of Chemical Ecology  2014;40:297-306.
Host plant resistance to insect attack and expansion of insect pests to novel hosts may to be modulated by phenolic compounds in host plants. Many studies have evaluated the role of phenolics in host plant resistance and the effect of phenolics on herbivore performance, but few studies have tested the joint effect of several compounds. Here, we used mixture-amount experimental design and response surface modeling to study the effects of a variety of phenolic compounds on the development and survival of Mexican fruit fly (Anastrepha ludens [Loew]), a notorious polyphagous pest of fruit crops that is likely to expand its distribution range under climate change scenarios. (+)- Catechin, phloridzin, rutin, chlorogenic acid, and p-coumaric acid were added individually or in mixtures at different concentrations to a laboratory diet used to rear individuals of A. ludens. No effect was observed with any mixture or concentration on percent pupation, pupal weight, adult emergence, or survival from neonate larvae to adults. Larval weight, larval and pupal developmental time, and the prevalence of adult deformities were affected by particular mixtures and concentrations of the compounds tested. We suggest that some combinations/concentrations of phenolic compounds could contribute to the management of A. ludens. We also highlight the importance of testing mixtures of plant secondary compounds when exploring their effects upon insect herbivore performance, and we show that mixture-amount design is a useful tool for this type of experiments.
Electronic supplementary material
The online version of this article (doi:10.1007/s10886-014-0404-6) contains supplementary material, which is available to authorized users.
PMCID: PMC3972442  PMID: 24619732
Anastrepha ludens; Larval performance; Phenolic compounds; Response-surface modeling; Secondary compounds; Tephritidae; Diptera
3.  Disrupting hedgehog and WNT signaling interactions promotes cleft lip pathogenesis 
The Journal of Clinical Investigation  2014;124(4):1660-1671.
Cleft lip, which results from impaired facial process growth and fusion, is one of the most common craniofacial birth defects. Many genes are known to be involved in the etiology of this disorder; however, our understanding of cleft lip pathogenesis remains incomplete. In the present study, we uncovered a role for sonic hedgehog (SHH) signaling during lip fusion. Mice carrying compound mutations in hedgehog acyltransferase (Hhat) and patched1 (Ptch1) exhibited perturbations in the SHH gradient during frontonasal development, which led to hypoplastic nasal process outgrowth, epithelial seam persistence, and cleft lip. Further investigation revealed that enhanced SHH signaling restricts canonical WNT signaling in the lambdoidal region by promoting expression of genes encoding WNT inhibitors. Moreover, reduction of canonical WNT signaling perturbed p63/interferon regulatory factor 6 (p63/IRF6) signaling, resulting in increased proliferation and decreased cell death, which was followed by persistence of the epithelial seam and cleft lip. Consistent with our results, mutations in genes that disrupt SHH and WNT signaling have been identified in both mice and humans with cleft lip. Collectively, our data illustrate that altered SHH signaling contributes to the etiology and pathogenesis of cleft lip through antagonistic interactions with other gene regulatory networks, including the canonical WNT and p63/IRF6 signaling pathways.
PMCID: PMC3973078  PMID: 24590292
4.  Relationship between Trough Plasma and Epithelial Lining Fluid Concentrations of Voriconazole in Lung Transplant Recipients 
Trough (predose) voriconazole concentrations in plasma and pulmonary epithelial lining fluid (ELF) of lung transplant recipients receiving oral voriconazole preemptive treatment were determined. The mean (± standard deviation [SD]) ELF/plasma ratio was 12.5 ± 6.3. A strong positive linear relationship was noted between trough plasma and ELF voriconazole concentrations (r2 = 0.87), suggesting the feasibility of using trough plasma voriconazole concentration as a surrogate to estimate the corresponding concentration in ELF of lung transplant recipients.
PMCID: PMC3754345  PMID: 23817382
5.  Pilot study of the impact sacroiliac joint manipulation has on walking kinematics using motion analysis technology 
Journal of Chiropractic Medicine  2013;12(3):143-152.
The purpose of this study was to evaluate the feasibility of engaging in a series of larger studies measuring the effect of sacroiliac joint manipulation on walking kinematics using motion analysis technology.
Twelve college students engaged in a baseline 90-second gait analysis at 1.5 mph using infrared VICON cameras. Following this, they underwent a prone heel comparison test for functional leg length inequality. Upon examination, participants were then classified as follows: left short leg, right short leg, or no short leg. Participants in each of the 2 short leg branches of this study were then randomized to receive either chiropractic manipulative therapy to the posterior superior iliac spine on the short limb side or no manipulation. Recruitment was ongoing for this pilot study until 1 participant was recruited in each of the following 5 comparative study groups: left short leg—manipulation, left short leg—no manipulation (control 1), right short leg—manipulation, right short leg—no manipulation (control 2), and no short leg (control 3). All participants then underwent another 90-second gait analysis. Data were then grouped and submitted to a blinded biomechanist to determine if there were any unique biomechanical differences between the groups.
No statistically significant differences were measured because of this being a pilot study with a small sample size.
The data from this study indicate that a series of larger studies with this design is feasible.
PMCID: PMC3838716  PMID: 24396314
Manipulation, chiropractic; Gait; Biomechanics; Locomotion
6.  Distinct Requirements for Cranial Ectoderm and Mesenchyme-Derived Wnts in Specification and Differentiation of Osteoblast and Dermal Progenitors 
PLoS Genetics  2014;10(2):e1004152.
The cranial bones and dermis differentiate from mesenchyme beneath the surface ectoderm. Fate selection in cranial mesenchyme requires the canonical Wnt effector molecule β-catenin, but the relative contribution of Wnt ligand sources in this process remains unknown. Here we show Wnt ligands are expressed in cranial surface ectoderm and underlying supraorbital mesenchyme during dermal and osteoblast fate selection. Using conditional genetics, we eliminate secretion of all Wnt ligands from cranial surface ectoderm or undifferentiated mesenchyme, to uncover distinct roles for ectoderm- and mesenchyme-derived Wnts. Ectoderm Wnt ligands induce osteoblast and dermal fibroblast progenitor specification while initiating expression of a subset of mesenchymal Wnts. Mesenchyme Wnt ligands are subsequently essential during differentiation of dermal and osteoblast progenitors. Finally, ectoderm-derived Wnt ligands provide an inductive cue to the cranial mesenchyme for the fate selection of dermal fibroblast and osteoblast lineages. Thus two sources of Wnt ligands perform distinct functions during osteoblast and dermal fibroblast formation.
Author Summary
Craniofacial abnormalities are relatively common congenital birth defects, and the Wnt signaling pathway and its effectors have key roles in craniofacial development. Wntless/Gpr177 is required for the efficient secretion of all Wnt ligands and maps to a region that contains SNPs strongly associated with reduced bone mass, and heterozygous deletion is associated with facial dysmorphology. Here we test the role of specific sources of secreted Wnt proteins during early stages of craniofacial development and obtained dramatic craniofacial anomalies. We found that the overlying cranial surface ectoderm Wnts generate an instructive cue of Wnt signaling for skull bone and skin cell fate selection and transcription of additional Wnts in the underlying mesenchyme. Once initiated, mesenchymal Wnts may maintain Wnt signal transduction and function in an autocrine manner during differentiation of skull bones and skin. These results highlight how Wnt ligands from two specific tissue sources are integrated for normal craniofacial patterning and can contribute to complex craniofacial abnormalities.
PMCID: PMC3930509  PMID: 24586192
7.  Efficacy and non-target impact of spinosad, Bti and temephos larvicides for control of Anopheles spp. in an endemic malaria region of southern Mexico 
Parasites & Vectors  2014;7:55.
The larvicidal efficacy of the naturally derived insecticide spinosad, for control of immature stages of Anopheles albimanus and associated culicids, was compared to that of synthetic and biological larvicides. Effects on non-target insects were also determined.
A field trial was performed in replicated temporary pools during the rainy season, in southern Mexico. Pools were treated with 10 ppm a.i. spinosad (Tracer 480SC), Bti granules applied at 2 kg/ha (VectoBac WDG, ABG-6511), and 100 ml/ha temephos (50 EC), or an untreated control. Numbers of immature mosquitoes, and aquatic insects in pools were monitored for 20 weeks.
Samples of immature mosquitoes comprised approximately 10% An. albimanus, 70% Culex spp. (mostly Cx. melanoconion and Cx. coronator) and 20% Uranotaenia lowii. The most effective larvicides were spinosad and temephos that eliminated An. albimanus in 16 out of 20 post-treatment samples, or 9 weeks of continuous control of immature stages, respectively. These larvicides resulted in 15 and 5 weeks of elimination of Culex spp., respectively, or 20 and 4 weeks of continuous elimination of U. lowii, respectively. Bti treatment provided little consistent control. Aquatic insects were recorded comprising 3 orders, 20 families, 40 genera and 44 species. Shannon diversity index values (H’) for aquatic insects were highest in the control (0.997) and Bti (0.974) treatments, intermediate in the spinosad treatment (0.638) and lowest in the temephos treatment (0.520). Severely affected non-target insects in the spinosad and temephos treated pools were predatory Coleoptera, Hemiptera and Odonata, which in the case of spinosad was likely due to the high concentration applied. Bti had little effect on aquatic insects.
The spinosad treatment retained larvicidal activity for markedly longer than expected. Spinosad is likely to be an effective tool for control of anopheline and other pool-breeding mosquitoes in tropical regions. Non-target effects of spinosad on aquatic insects merit further study, but were likely related to the concentration of the product used.
PMCID: PMC3915226  PMID: 24479683
8.  Diversity of mosquitoes and the aquatic insects associated with their oviposition sites along the Pacific coast of Mexico 
Parasites & Vectors  2014;7:41.
The abundance, richness and diversity of mosquitoes and aquatic insects associated with their oviposition sites were surveyed along eight states of the Pacific coast of Mexico. Diversity was estimated using the Shannon index (H’), similarity measures and cluster analysis.
Oviposition sites were sampled during 2–3 months per year, over a three year period. Field collected larvae and pupae were reared and identified to species following adult emergence. Aquatic insects present at oviposition sites were also collected, counted and identified to species or genus.
In total, 15 genera and 74 species of mosquitoes were identified: Anopheles pseudopunctipennis, An. albimanus and Aedes aegypti were the most abundant and widely-distributed species, representing 47% of total mosquito individuals sampled. New species records for certain states are reported. Anopheline diversity was lowest in Sinaloa state (H’ = 0.54) and highest in Chiapas (H’ = 1.61) and Michoacán (H’ = 1.56), whereas culicid diversity was lowest in Michoacán (H’ = 1.93), Colima (H’ = 1.95), Sinaloa (H’ = 1.99) and Jalisco (H’ = 2.01) and highest in Chiapas (H’ = 2.66). In total, 10 orders, 57 families, 166 genera and 247 species of aquatic insects were identified in samples. Aquatic insect diversity was highest in Chiapas, Oaxaca and Michoacán (H’ = 3.60-3.75). Mosquito larval/pupal abundance was not correlated with that of predatory Coleoptera and Hemiptera.
This represents the first update on the diversity and geographic distribution of the mosquitoes and aquatic insects of Mexico in over five decades. This information has been cataloged in Mexico’s National Biodiversity Information System (SNIB-CONABIO) for public inspection.
PMCID: PMC3923424  PMID: 24450800
9.  Correction: MicroRNA-17-92, a Direct Ap-2α Transcriptional Target, Modulates T-Box Factor Activity in Orofacial Clefting 
PLoS Genetics  2013;9(12):10.1371/annotation/90602bc3-5052-49ac-a7fb-33210d7c8b4d.
PMCID: PMC3867558
10.  Expression of a Peroral Infection Factor Determines Pathogenicity and Population Structure in an Insect Virus 
PLoS ONE  2013;8(11):e78834.
A Nicaraguan isolate of Spodoptera frugiperda multiple nucleopolyhedrovirus is being studied as a possible biological insecticide. This virus exists as a mixture of complete and deletion genotypes; the latter depend on the former for the production of an essential per os transmission factor (pif1) in coinfected cells. We hypothesized that the virus population was structured to account for the prevalence of pif1 defector genotypes, so that increasing the abundance of pif1 produced by a cooperator genotype in infected cells would favor an increased prevalence of the defector genotype. We tested this hypothesis using recombinant viruses with pif1 expression reprogrammed at its native locus using two exogenous promoters (egt, p10) in the pif2/pif1 intergenic region. Reprogrammed viruses killed their hosts markedly faster than the wild-type and rescue viruses, possibly due to an earlier onset of systemic infection. Group success (transmission) depended on expression of pif1, but overexpression was prejudicial to group-specific transmissibility, both in terms of reduced pathogenicity and reduced production of virus progeny from each infected insect. The presence of pif1-overproducing genotypes in the population was predicted to favor a shift in the prevalence of defector genotypes lacking pif1-expressing capabilities, to compensate for the modification in pif1 availability at the population level. As a result, defectors increased the overall pathogenicity of the virus population by diluting pif1 produced by overexpressing genotypes. These results offer a new and unexpected perspective on cooperative behavior between viral genomes in response to the abundance of an essential public good that is detrimental in excess.
PMCID: PMC3818493  PMID: 24223853
11.  Ectodermal-derived Endothelin1 is required for patterning the distal and intermediate domains of the mouse mandibular arch 
Developmental biology  2012;371(1):47-56.
Morphogenesis of the vertebrate head relies on proper dorsal-ventral (D-V) patterning of neural crest cells (NCC) within the pharyngeal arches. Endothelin-1 (Edn1)-induced signaling through the endothelin-A receptor (Ednra) is crucial for cranial NCC patterning within the mandibular portion of the first pharyngeal arch, from which the lower jaw arises. Deletion of Edn1, Ednra or endothelin-converting enzyme in mice causes perinatal lethality due to severe craniofacial birth defects. These include homeotic transformation of mandibular arch-derived structures into more maxillary-like structures, indicating a loss of NCC identity. All cranial NCCs express Ednra whereas Edn1 expression is limited to the overlying ectoderm, core paraxial mesoderm and pharyngeal pouch endoderm of the mandibular arch as well as more caudal arches. To define the developmental significance of Edn1 from each of these layers, we used Cre/loxP technology to inactivate Edn1 in a tissue-specific manner. We show that deletion of Edn1 in either the mesoderm or endoderm alone does not result in cellular or molecular changes in craniofacial development. However, ectodermal deletion of Edn1 results in craniofacial defects with concomitant changes in the expression of early mandibular arch patterning genes. Importantly, our results also both define for the first time in mice an intermediate mandibular arch domain similar to the one defined in zebrafish and show that this region is most sensitive to loss of Edn1. Together, our results illustrate an integral role for ectoderm-derived Edn1 in early arch morphogenesis, particularly in the intermediate domain.
PMCID: PMC3470875  PMID: 22902530
neural crest cell; conditional knockout; mouse; endothelin; craniofacial
12.  The sf32 Unique Gene of Spodoptera frugiperda Multiple Nucleopolyhedrovirus (SfMNPV) Is a Non-Essential Gene That Could Be Involved in Nucleocapsid Organization in Occlusion-Derived Virions 
PLoS ONE  2013;8(10):e77683.
A recombinant virus lacking the sf32 gene (Sf32null), unique to the Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV), was generated by homologous recombination from a bacmid comprising the complete viral genome (Sfbac). Transcriptional analysis revealed that sf32 is an early gene. Occlusion bodies (OBs) of Sf32null contained 62% more genomic DNA than viruses containing the sf32 gene, Sfbac and Sf32null-repair, although Sf32null DNA was three-fold less infective when injected in vivo. Sf32null OBs were 18% larger in diameter and contained 17% more nucleocapsids within ODVs than those of Sfbac. No significant differences were detected in OB pathogenicity (50% lethal concentration), speed-of-kill or budded virus production in vivo. In contrast, the production of OBs/larva was reduced by 39% in insects infected by Sf32null compared to those infected by Sfbac. The SF32 predicted protein sequence showed homology (25% identity, 44% similarity) to two adhesion proteins from Streptococcus pyogenes and a single N-mirystoylation site was predicted. We conclude that SF32 is a non-essential protein that could be involved in nucleocapsid organization during ODV assembly and occlusion, resulting in increased numbers of nucleocapsids within ODVs.
PMCID: PMC3813766  PMID: 24204916
13.  Complete Genome Sequences of Five Chrysodeixis chalcites Nucleopolyhedrovirus Genotypes from a Canary Islands Isolate 
Genome Announcements  2013;1(5):e00873-13.
The Chrysodeixis chalcites single nucleopolyhedrovirus (ChchSNPV) infects and kills C. chalcites larvae, an important pest of banana crops in the Canary Islands. Five genotypes present in the most prevalent and widespread isolate in the Canary Islands were sequenced, providing genetic data relevant to the genotypic and phenotypic diversity of this virus.
PMCID: PMC3813185  PMID: 24158555
14.  Deletion Genotypes Reduce Occlusion Body Potency but Increase Occlusion Body Production in a Colombian Spodoptera frugiperda Nucleopolyhedrovirus Population 
PLoS ONE  2013;8(10):e77271.
A Colombian field isolate (SfCOL-wt) of Spodoptera frugiperda multiple nucleopolyhedrovirus (SfMNPV) is a mixture of different genotypes. To evaluate the insecticidal properties of the different genotypic variants, 83 plaque purified virus were characterized. Ten distinct genotypes were identified (named A through J). SfCOL-A was the most prevalent (71±2%; mean ± SE) showing a PstI restriction profile indistinguishable to that of SfCOL-wt. The remaining nine genotypes presented genomic deletions of 3.8 - 21.8 Kb located mainly between nucleotides 11,436 and 33,883 in the reference genome SfMNPV-B, affecting the region between open reading frames (ORFs) sf20 and sf33. The insecticidal activity of each genotype from SfCOL-wt and several mixtures of genotypes was compared to that of SfCOL-wt. The potency of SfCOL-A occlusion bodies (OBs) was 4.4-fold higher than SfCOL-wt OBs, whereas the speed of kill of SfCOL-A was similar to that of SfCOL-wt. Deletion genotype OBs were similarly or less potent than SfCOL-wt but six deletion genotypes were faster killing than SfCOL-wt. The potency of genotype mixtures co-occluded within OBs were consistently reduced in two-genotype mixtures involving equal proportions of SfCOL-A and one of three deletion genotypes (SfCOL-C, -D or -F). Speed of kill and OB production were improved only when the certain genotype mixtures were co-occluded, although OB production was higher in the SfCOL-wt isolate than in any of the component genotypes, or mixtures thereof. Deleted genotypes reduced OB potency but increased OB production of the SfCOL-wt population, which is structured to maximize the production of OBs in each infected host.
PMCID: PMC3792916  PMID: 24116220
15.  MicroRNA-17-92, a Direct Ap-2α Transcriptional Target, Modulates T-Box Factor Activity in Orofacial Clefting 
PLoS Genetics  2013;9(9):e1003785.
Among the most common human congenital anomalies, cleft lip and palate (CL/P) affects up to 1 in 700 live births. MicroRNA (miR)s are small, non-coding RNAs that repress gene expression post-transcriptionally. The miR-17-92 cluster encodes six miRs that have been implicated in human cancers and heart development. We discovered that miR-17-92 mutant embryos had severe craniofacial phenotypes, including incompletely penetrant CL/P and mandibular hypoplasia. Embryos that were compound mutant for miR-17-92 and the related miR-106b-25 cluster had completely penetrant CL/P. Expression of Tbx1 and Tbx3, the DiGeorge/velo-cardio-facial (DGS) and Ulnar-mammary syndrome (UMS) disease genes, was expanded in miR-17-92 mutant craniofacial structures. Both Tbx1 and Tbx3 had functional miR seed sequences that mediated gene repression. Analysis of miR-17-92 regulatory regions uncovered conserved and functional AP-2α recognition elements that directed miR-17-92 expression. Together, our data indicate that miR-17-92 modulates expression of critical T-box transcriptional regulators during midface development and is itself a target of Bmp-signaling and the craniofacial pioneer factor AP-2α. Our data are the first genetic evidence that an individual miR or miR cluster is functionally important in mammalian CL/P.
Author Summary
CL/P are very common birth defects in humans. The genetic mechanism underlying CL/P pathogenesis is poorly understood. MiRs, small non-coding RNAs that function to post-transcriptionally regulate gene expression, have been identified as pivotal modulators of various developmental events and diseases. To date, there is no individual miR or miR cluster that has been identified as functionally essential in mammalian CL/P. Here, we have discovered that deletion of miR-17-92 cluster in mice results in craniofacial malformations including CL/P. Importantly, MIR-17-92 is located on a critical human chromosome region associated with 13q deletion syndrome, a chromosomal disorder that presents with defects including CL/P, suggesting the advantages of our animal model to study human disease. Moreover, our work demonstrated that miR-17-92 cluster directly repressed T-box factors, which have critical functions during craniofacial development. We further showed that miR-17-92 was directly activated by Bmp-signaling and transcription factor AP-2α. Together, our work identified a novel miR-mediated transcriptional network underlying CL/P, providing new insights into craniofacial developmental biology.
PMCID: PMC3777996  PMID: 24068957
16.  Gender-Mediated Differences in Vertical Transmission of a Nucleopolyhedrovirus 
PLoS ONE  2013;8(8):e70932.
With the development of sensitive molecular techniques for detection of low levels of asymptomatic pathogens, it becoming clear that vertical transmission is a common feature of some insect pathogenic viruses, and likely to be essential to virus survival when opportunities for horizontal transmission are unfavorable. Vertical transmission of Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) is common in natural populations of S. exigua. To assess whether gender affected transgenerational virus transmission, four mating group treatments were performed using healthy and sublethally infected insects: i) healthy males (H♂)×healthy females (H♀); ii) infected males (I♂)×healthy females (H♀); iii) healthy males (H♂)×infected females (I♀) and iv) infected males (I♂)×infected females (I♀). Experimental adults and their offspring were analyzed by qPCR to determine the prevalence of infection. Both males and females were able to transmit the infection to the next generation, although female-mediated transmission resulted in a higher prevalence of infected offspring. Male-mediated venereal transmission was half as efficient as maternally-mediated transmission. Egg surface decontamination studies indicated that the main route of transmission is likely transovarial rather than transovum. Both male and female offspring were infected by their parents in similar proportions. Incorporating vertically-transmitted genotypes into virus-based insecticides could provide moderate levels of transgenerational pest control, thereby extending the periods between bioinsecticide applications.
PMCID: PMC3733637  PMID: 23940671
17.  Analagous Population Structures for Two Alphabaculoviruses Highlight a Functional Role for Deletion Mutants 
A natural Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) isolate from Florida shares a strikingly similar genotypic composition to that of a natural Spodoptera frugiperda MNPV (SfMNPV) isolate from Nicaragua. Both isolates comprise a high proportion of large-deletion genotypes that lack genes that are essential for viral replication or transmission. To determine the likely origins of such genotypically similar population structures, we performed genomic and functional analyses of these genotypes. The homology of nucleotides in the deleted regions was as high as 79%, similar to those of other colinear genomic regions, although some SfMNPV genes were not present in SeMNPV. In addition, no potential consensus sequences were shared between the deletion flanking sequences. These results indicate an evolutionary mechanism that independently generates and sustains deletion mutants within each virus population. Functional analyses using different proportions of complete and deletion genotypes were performed with the two viruses in mixtures of occlusion bodies (OBs) or co-occluded virions. Ratios greater than 3:1 of complete/deletion genotypes resulted in reduced pathogenicity (expressed as median lethal dose), but there were no significant changes in the speed of kill. In contrast, OB yields increased only in the 1:1 mixture. The three phenotypic traits analyzed provide a broader picture of the functional significance of the most extensively deleted SeMNPV genotype and contribute toward the elucidation of the role of such mutants in baculovirus populations.
PMCID: PMC3568584  PMID: 23204420
18.  Overlapping Expression Patterns and Redundant Roles for AP-2 Transcription Factors in the Developing Mammalian Retina 
We have previously shown that the transcription factor AP-2α (Tcfap2a) is expressed in postmitotic developing amacrine cells in the mouse retina. Although retina-specific deletion of Tcfap2a did not affect retinogenesis, two other family members, AP-2β and AP-2γ, showed expression patterns similar to AP-2α.
Here we show that, in addition to their highly overlapping expression patterns in amacrine cells, AP-2α and AP-2β are also co-expressed in developing horizontal cells. AP-2γ expression is restricted to amacrine cells, in a subset that is partially distinct from the AP-2α/β-immunopositive population. To address possible redundant roles for AP-2α and AP-2β during retinogenesis, Tcfap2a/b-deficient retinas were examined. These double mutants showed a striking loss of horizontal cells and an altered staining pattern in amacrine cells that were not detected upon deletion of either family member alone.
These studies have uncovered critical roles for AP-2 activity in retinogenesis, delineating the overlapping expression patterns of Tcfap2a, Tcfap2b, and Tcfap2c in the neural retina, and revealing a redundant requirement for Tcfap2a and Tcfap2b in horizontal and amacrine cell development.
PMCID: PMC3700368  PMID: 22411557
AP-2 transcription factors; retinal development; horizontal cell; amacrine cell; Tcfap2a/b double mutant; redundant roles
19.  Time from symptoms to definitive diagnosis of idiopathic pulmonary arterial hypertension: The delay study 
Pulmonary Circulation  2013;3(1):89-94.
Survival rates for patients with idiopathic pulmonary arterial hypertension (IPAH) have improved with the introduction of PAH-specific therapies. However, the time between patient-reported onset of symptoms and a definitive diagnosis of IPAH is consistently delayed. We conducted a retrospective, multi-center, descriptive investigation in order to (a) understand what factors contribute to persistent diagnostic delays, and (b) examine the time from initial symptom onset to a definitive diagnosis of IPAH. Between January 2007 and December 2008, we enrolled consecutively diagnosed adults with IPAH from four tertiary referral centers in Australia. Screening of patient records and “one-on-one” interviews were used to determine the time from patient-described initial symptoms to a diagnosis of IPAH, confirmed by right heart catheterization (RHC). Thirty-two participants (69% female) were studied. Mean age at symptom onset was 56 ± 16.4 years and 96% reported exertional dyspnea. Mean time from symptom onset to diagnosis was 47 ± 34 months with patients subsequently aged 60 ± 17.3 years. Patients reported 5.3 ± 3.8 GP visits and 3.0 ± 2.1 specialist reviews before being seen at a pulmonary hypertension (PH) center. Advanced age, number of general practitioner (GP) visits, heart rate, and systolic blood pressure at the time of diagnosis were significantly associated with the observed delay. We found a significant delay of 3.9 years from symptom onset to a diagnosis of IPAH in Australia. Exertional dyspnea is the most common presenting symptom. Current practice within Australia does not appear to have the specific capacity for timely, multi-factorial evaluation of breathlessness and potential IPAH.
PMCID: PMC3641745  PMID: 23662179
diagnosis; epidemiology; pulmonary hypertension; pulmonary arterial hypertension
20.  Vgll2a is required for neural crest cell survival during zebrafish craniofacial development 
Developmental biology  2011;357(1):269-281.
Invertebrate and vertebrate vestigial (vg) and vestigial-like (vgl) genes are involved in embryonic patterning and cell fate determination. These genes encode cofactors that interact with members of the TEAD/Scalloped family of transcription factors and modulate their activity. We have previously shown that, in mice, Vgll2 is differentially expressed in the developing facial prominences. In this study, we show that the zebrafish ortholog vgll2a is expressed in the pharyngeal endoderm and ectoderm surrounding the neural crest derived mesenchyme of the pharyngeal arches. Moreover, both the FGF and retinoic acid (RA) signaling pathways, which are critical components of the hierarchy controlling craniofacial patterning, regulate this domain of vgll2a expression. Consistent with these observations, vgll2a is required within the pharyngeal endoderm for NCC survival and pharyngeal cartilage development. Specifically, knockdown of Vgll2a in zebrafish embryos using Morpholino injection results in increased cell death within the pharyngeal arches, aberrant endodermal pouch morphogenesis, and hypoplastic cranial cartilages. Overall, our data reveal a novel non-cell autonomous role for Vgll2a in development of the NCC-derived vertebrate craniofacial skeleton.
PMCID: PMC3519931  PMID: 21741961
Vestigial-like; Vgl-2; VITO-1; craniofacial; zebrafish; FGF; Retinoic Acid; Cell Death
21.  Activation of the Hedgehog Signaling Pathway in the Developing Lens Stimulates Ectopic FoxE3 Expression and Disruption in Fiber Cell Differentiation 
The signaling pathways and transcriptional effectors responsible for directing mammalian lens development provide key regulatory molecules that can inform our understanding of human eye defects. The hedgehog genes encode extracellular signaling proteins responsible for patterning and tissue formation during embryogenesis. Signal transduction of this pathway is mediated through activation of the transmembrane proteins smoothened and patched, stimulating downstream signaling resulting in the activation or repression of hedgehog target genes. Hedgehog signaling is implicated in eye development, and defects in hedgehog signaling components have been shown to result in defects of the retina, iris, and lens.
We assessed the consequences of constitutive hedgehog signaling in the developing mouse lens using Cre-LoxP technology to express the conditional M2 smoothened allele in the embryonic head and lens ectoderm.
Although initial lens development appeared normal, morphological defects were apparent by E12.5 and became more significant at later stages of embryogenesis. Altered lens morphology correlated with ectopic expression of FoxE3, which encodes a critical gene required for human and mouse lens development. Later, inappropriate expression of the epithelial marker Pax6, and as well as fiber cell markers c-maf and Prox1 also occurred, indicating a failure of appropriate lens fiber cell differentiation accompanied by altered lens cell proliferation and cell death.
Our findings demonstrate that the ectopic activation of downstream effectors of the hedgehog signaling pathway in the mouse lens disrupts normal fiber cell differentiation by a mechanism consistent with a sustained epithelial cellular developmental program driven by FoxE3.
This study shows the ability of the lens to respond to altered Smoothened activity. Constitutive activation of Smoothened in the surface ectoderm and derivatives, including the lens results in altered expression of lens markers and abnormal lens differentiation and morphology.
PMCID: PMC3385968  PMID: 22491411
22.  A Conserved Pbx-Wnt-p63-Irf6 Regulatory Module Controls Face Morphogenesis by Promoting Epithelial Apoptosis 
Developmental cell  2011;21(4):627-641.
Morphogenesis of mammalian facial processes requires coordination of cellular proliferation, migration, and apoptosis to develop intricate features. Cleft lip and/or palate (CL/P), the most frequent human craniofacial birth defect, can be caused by perturbation of any of these programs. Mutations of WNT, P63, and IRF6 yield CL/P in humans and mice; however, how these genes are regulated remains elusive. We generated mouse lines lacking Pbx genes in cephalic ectoderm and demonstrated that they exhibit fully penetrant CL/P and perturbed Wnt signalling. We also characterized a midfacial regulatory element that Pbx proteins bind in order to control the expression of Wnt9b-Wnt3, which in turn regulate p63. Altogether, we establish a Pbx-dependent Wnt-p63-Irf6 regulatory module in midfacial ectoderm that is conserved within mammals. Dysregulation of this network leads to localized suppression of midfacial apoptosis and CL/P. Ectopic Wnt ectodermal expression in Pbx mutants rescues the clefting, opening avenues for tissue repair.
PMCID: PMC3199312  PMID: 21982646
Face; Ectoderm; Lambdoidal Junction; Pbx; Wnt; p63; Irf6; Wnt midfacial enhancer; CL/P; Mouse
23.  Mutations in Hedgehog Acyltransferase (Hhat) Perturb Hedgehog Signaling, Resulting in Severe Acrania-Holoprosencephaly-Agnathia Craniofacial Defects 
PLoS Genetics  2012;8(10):e1002927.
Holoprosencephaly (HPE) is a failure of the forebrain to bifurcate and is the most common structural malformation of the embryonic brain. Mutations in SHH underlie most familial (17%) cases of HPE; and, consistent with this, Shh is expressed in midline embryonic cells and tissues and their derivatives that are affected in HPE. It has long been recognized that a graded series of facial anomalies occurs within the clinical spectrum of HPE, as HPE is often found in patients together with other malformations such as acrania, anencephaly, and agnathia. However, it is not known if these phenotypes arise through a common etiology and pathogenesis. Here we demonstrate for the first time using mouse models that Hedgehog acyltransferase (Hhat) loss-of-function leads to holoprosencephaly together with acrania and agnathia, which mimics the severe condition observed in humans. Hhat is required for post-translational palmitoylation of Hedgehog (Hh) proteins; and, in the absence of Hhat, Hh secretion from producing cells is diminished. We show through downregulation of the Hh receptor Ptch1 that loss of Hhat perturbs long-range Hh signaling, which in turn disrupts Fgf, Bmp and Erk signaling. Collectively, this leads to abnormal patterning and extensive apoptosis within the craniofacial primordial, together with defects in cartilage and bone differentiation. Therefore our work shows that Hhat loss-of-function underscrores HPE; but more importantly it provides a mechanism for the co-occurrence of acrania, holoprosencephaly, and agnathia. Future genetic studies should include HHAT as a potential candidate in the etiology and pathogenesis of HPE and its associated disorders.
Authors Summary
Craniofacial anomalies account for approximately one third of all birth defects, and holoprosencephaly (HPE) is the most common structural malformation of the embryonic brain. HPE is a failure of the forebrain to bifurcate and is a heterogeneous disorder that is often found in patients together with other craniofacial malformations. Currently, it is not known if these phenotypes arise through a common etiology and pathogenesis, as the genetic lesions responsible for HPE have only been identified in about 20% of affected individuals. Here we demonstrate for the first time that Hedgehog acyltransferase (Hhat) loss-of-function leads to holoprosencephaly together with acrania and agnathia, which highlights the importance of Hh signaling in complex craniofacial disorders.
PMCID: PMC3464201  PMID: 23055936
24.  Spinosad: a biorational mosquito larvicide for use in car tires in southern Mexico 
Parasites & Vectors  2012;5:95.
Car tires are important habitats for mosquito development because of the high density populations they can harbor and their presence in urban settings. Water in experimental tires was treated with one of three insecticides or an untreated control. Aquatic invertebrates were sampled at weekly intervals. Eggs, larval and pupal samples were laboratory-reared to estimate seasonal fluctuations in Aedes aegypti and Ae. albopictus abundance.
Spinosad treatments at 1 or 5 ppm (mg a.i./liter) provided 6–8 weeks of effective control of Ae. aegypti, Ae. albopictus, Culex quinquefasiatus and Cx. coronator larvae, both in the dry season and the rainy season when mosquito populations increased markedly in southern Mexico. Spinosad continued to provide partial control of larvae for several weeks after initial recolonization of treated tires. The larvicidal performance of VectoBac 12AS (Bacillus thuringiensis var. israelensis) was relatively poor with one week of complete control of Aedes spp. larvae and no discernible control of Culex spp., whereas the duration of larvicidal activity of 1% temephos mineral-based granules was intermediate between those of VectoBac and spinosad treatments. Populations of chironomids, ostracods and Toxorhynchites theobaldi were generally reduced in spinosad and temephos treatments, but were similar in control and VectoBac treatments.
The present study is the first to report spinosad as an effective larvicide against Cx. coronator, which is currently invading the southern United States. These results substantiate the use of spinosad as a highly effective mosquito larvicide, even in habitats such as unused car tires that can represent prolific sources of adult mosquitoes.
PMCID: PMC3407511  PMID: 22608138
25.  Epibranchial placode-derived neurons produce BDNF required for early sensory neuron development 
In mice, BDNF provided by the developing taste epithelium is required for gustatory neuron survival following target innervation. However, we find that expression of BDNF, as detected by BDNF-driven β-galactosidase, begins in the cranial ganglia before its expression in the central (hindbrain) or peripheral (taste papillae) targets of these sensory neurons, and before gustatory ganglion cells innervate either target. To test early BDNF function, we examined the ganglia of bdnf null mice before target innervation, and found that while initial neuron survival is unaltered, early neuron development is disrupted. In addition, fate mapping analysis in mice demonstrates that murine cranial ganglia arise from 2 embryonic populations, i.e., epibranchial placodes and neural crest, as has been described for these ganglia in non-mammalian vertebrates. Only placodal neurons produce BDNF, however, which indicates that prior to innervation, early ganglionic BDNF produced by placode-derived cells promotes gustatory neuron development.
PMCID: PMC3070660  PMID: 21246648
BDNF; cranial ganglia; maturation; gustatory; placode; neural crest; neuron; mouse; cre recombinase; fate mapping; ROSA reporter line

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