Aedes aegypti, an indoor day-biting mosquito. The most effective proven method for disease prevention has been by vector control by various chemical or biological means. Unfortunately insecticide resistance has already been observed on the Island and recently showed to significantly reduce the efficacy of vector control interventions. In this study, we investigated the distribution of resistance and the underlying mechanisms in nine Ae. aegypti populations. Statistical multifactorial approach was used to investigate the correlations between insecticide resistance levels, associated mechanisms and environmental factors characterizing the mosquito populations. Bioassays revealed high levels of resistance to temephos and deltamethrin and susceptibility to Bti in the 9 populations tested. Biochemical assays showed elevated detoxification enzyme activities of monooxygenases, carboxylesterases and glutathione S-tranferases in most of the populations. Molecular screening for common insecticide target-site mutations, revealed the presence of the “knock-down resistance” V1016I Kdr mutation at high frequency (>87%). Real time quantitative RT-PCR showed the potential involvement of several candidate detoxification genes in insecticide resistance. Principal Component Analysis (PCA) performed with variables characterizing Ae. aegypti from Martinique permitted to underline potential links existing between resistance distribution and other variables such as agriculture practices, vector control interventions and urbanization. Insecticide resistance is widespread but not homogeneously distributed across Martinique. The influence of environmental and operational factors on the evolution of the resistance and mechanisms are discussed.Dengue is an important mosquito borne viral disease in Martinique Island (French West Indies). The viruses responsible for dengue are transmitted by  Aedes aegypti remains the most effective solution to prevent dengue transmission. Environmental management, educational programs and mechanic elimination of the breeding habitats are continuously implemented but currently, the use of chemical and biological agents are the main methods for reducing the incidence of the disease.Dengue fever is a major public health problem in Martinique Island (French-West-Indies) and occurs in an endemo-epidemic pattern Ae. aegyptiKdr) mutation (V1016I) in this Martinique population which confers resistance to DDT and PYRs Unfortunately, vector control programs are facing operational challenges with the emergence and development of insecticide resistance in dengue vectors, especially Bacillus thuringiensis var israelensis  is the only insecticide used for such application. Space spraying treatments with vehicle-mounted or portable thermal fogger  are implemented during inter-epidemic periods  and during outbreaks to rapidly kill infected adult mosquitoes The massive use of different insecticide families for vector control since the 1950s has probably contributed to select for insecticide resistance in mosquitoes Ae. aegypti populations collected in several ecological settings in Martinique island. A statistical multifactorial approach was adopted to investigate the possible relationship between resistance levels, associated mechanisms and environmental factors such as agriculture, pollution and urbanization in Martinique populations.In this study we investigated the insecticide resistance level and the associated molecular mechanisms in nine Ae. aegypti was collected from individual houses as larvae or pupae in nine localities of Martinique in February 2009 (Two laboratory strains from French Polynesia (Bora-Bora) and Benin (SBE) and nine field-caught populations were used in the study. The two laboratory strains are susceptible to all insecticides and have been used as reference strains for resistance assays and as outgroups in order to gain comparative power and compensate for genetic diversity for gene expression analyses. ary 2009 . PopulatBti Vectobac®12AS . Bioassays were performed using late third and early fourth-instar larvae of each population. Four replicates per concentration and 5 to 8 concentrations in the activity range of each insecticide were used (n = 100 per replicate per concentration). Larval mortality was recorded after 24 h of insecticide exposure.Larval and adult bioassays were performed following WHO protocols Adult female mosquitoes of each population were used for the tarsal contact with treated filter papers in comparison with the Bora-Bora strain as described in Marcombe et al. 50 and LC95 for larvae) and knock-down time (KDT50 and KDT95 for adults) were calculated together with their 95% confidence intervals (95% CI). Adult mortality after 24 h exposure to deltamethrin was also recorded for each population. Populations from Martinique were considered as having different susceptibility to a given pesticide compared to the susceptible Bora-Bora strain when the ratio between their LC50/95 or KDT50/95 (resistance ratio: RR50/95) had confidence limits excluding the value of 1.Percent mortality was corrected using Abbott's formula P450 monooxygenases (P450s) level, carboxy/cholinesterases (CCEs) activities and Glutathione S-transferases (GSTs) activities were assayed from single 3 days-old F1 females (n = 47) according to the microplate methods described by Hemingway Kdr mutations have been described 5′ ACTACATCGAGAATGTGGATCG 3′) and Aed2A (5′ TTGTTGGTGTC GTTGTCGGCCGTCGG 3′) primers. This region covers exons 21 and 22 of the sodium channel gene allowing to detect the following Kdr mutations: I1011M, I1011V, V1016I and V1016G. After purification of the PCR products using the AMPure kit , the BigDye terminator v3.1 kit  was used with the same primers for sequencing. Sequence reactions were purified using the CleanSEQ kit (Agencourt) and were then sequenced on an ABI Prism 3130xl analyzer (Applied Biosystems). The SeqScape software was used for sequence analysis. Hardy-Weinberg equilibrium was tested using the exact probability test Total DNA of single female mosquitoes of each strain and Martinique populations (n = 32) was extracted using a CTAB protocol Ae. aegypti with dichlorvos and propoxur concentrations of 3.10−4 M and 4.10−4 M, respectively. The TDP test allows discriminating all possible phenotypes containing the G119S, F290V and wild-type (susceptible) alleles.The phenotypes of the acetylcholine esterase AChE1, encoded by the ace-1 gene, were investigated in each population (n = 24) using the previously described TDP test CYP genes), 2 P450-cofactors, the cytochrome-P450-reductase (CPR gene) and the cytochrome b5 (CytB5 gene), 1 CCE (CCEae3A) and 3 GSTs genes were measured by real-time quantitative RT-qPCR in larvae and adult females of the 2 susceptible strains and the 9 field-caught populations. These genes were chosen because of their putative involvement in metabolic resistance to chemical insecticides th stage larvae or thirty 4–6 days-old adult females were collected alive for RNA extraction using Trizol (Invitrogene). RNA extractions, reverse transcriptions and quantitative PCR analyses were performed as described in Marcombe et al T method taking into account PCR efficiency and using the genes encoding the ribosomal protein L8  and R7 (AeRPS7 GenBank accession number EAT38624.1) for normalization. Results were expressed as mean transcription ratios (±SE) between larvae or adults of the different populations and the susceptible Bora-Bora strain. Genes showing a transcription ratio higher than 2-fold and a P value<0.05 where considered significantly over-transcribed .Transcription levels of 6 P450s  across the nine populations of Martinique. Only candidate genes found over-transcribed in at least one population at any life stage were considered. The variables used and their respective ‘names’ were as follow: mean gene transcription ratio versus Bora-Bora strain (‘Gene_Lv’ for larvae or ‘Gene_Ad’ for adults), larval resistance to temephos (‘RR50 tem’), adult resistance to deltamethrin knock down effect (‘RR50 delta’), % adult surviving 24 h after deltamethrin exposure (‘Alive 24 h delta’), Kdr mutation frequency (‘V1016I’), insecticide pressure, agriculture and urbanization. Insecticides pressure was represented by two variables obtained from data provided by the vector control unit of Martinique. The first variable (‘Pulv’) represents the number of outdoor deltamethrin pulverization  applications made routinely between 2006 and 2009 in each population area. The second variable (‘Int’) represents the number of specific interventions because of high entomological indices or dengue cases between 2006 and 2009, corresponding to larval treatment with Bti or temephos and deltamethrin pulverizations . The environment was described by five variables chosen for their putative role in the selection of insecticide resistance: sugar cane cultures (‘Sug’), bananas cultures (‘Ban’), other agricultural crops (‘Agri’), organochlorine pollution . This percentage was obtained by pixel measurement using the software MESURIM . PCAs were performed using R software Potential relationship between insecticide resistance levels, gene transcription levels, 50 ranged from 13-fold (SJOS) to 36-fold (GMRN) and RR95 ranged from 26-fold (SJOS) to 153-fold (VCLN). WHO tube tests with 0.05% deltamethrin on adult mosquitoes showed resistance to knockdown effect with RR50 ranging from 3.71-fold for RSAL population to 6.71-fold for SAN population. Mortality after 24 h deltamethrin exposure ranged from only 19% for AJPB population to 90% for the RSAL population while mortality in the susceptible strains reached 100%. For Bti, all Martinique populations showed low RRs comparatively to the susceptible Bora-Bora strain (maximum RR50 of 2.26-fold), indicating that all populations tested were mostly susceptible to Bti.Bioassays showed that the nine populations of Martinique were resistant to temephos and deltamethrin in comparison with the laboratory susceptible Bora-Bora strain and the susceptible strain SBE originating from Benin . For temDetoxification enzyme activities were estimated for each population at the adult stage . For allKdr mutation at position 1016 (GTA to ATA) leading to the replacement of a valine by an isoleucine (V1016I) at a high allelic frequency for all of Martinique populations . No other Kdr resistant allele was detected in these populations. No Kdr mutation was found in the two susceptible strains Bora-Bora and SBE.Sequencing of the voltage-gated sodium channel gene from single mosquitoes revealed the presence of the All mosquito test populations from Martinique showed similar percentages of AChE inhibition with dichlorvos and propoxur compared to the susceptible Bora-Bora strain excepted for RSAL and VCLN (only for dichlorvos for this latter). However, the differences were not strong enough to suspect the presence of insensitive AChE in the populations tested .CYP6Z6 and CYP6Z8 were both over-transcribed in 6 populations of Martinique  compared to the susceptible strains .Transcription profiles of twelve candidate detoxification genes potentially involved in metabolic resistance to insecticides were compared between susceptible strains and Martinique test populations at the larval and adult stages. Genes with transcription ratio over 2-fold and a P value<0.05 were considered significantly over-transcribed. In larvae , the P45 strains . The gener genes , the P45CYP6Z6 and CYP6Z8 were over-transcribed in SPIER, LAM, RSAL, SAN and AJPB, GRMN, SJOS, LAM, FDF, VCLN respectively. CYP9J22 was over-transcribed in all Martinique populations except FDF while CYP9M9 was over-transcribed in AJPB, GRMN, SJOS, FDF and SAN. No significant differences of transcription level were observed for CYP6M6 and CYP6M11 compared to the susceptible strains. Among the two P450-cofactor genes, only the CPR was over-transcribed in the GRMN, SJOS, FDF, RSAL and SAN populations .In adults, four different P450 genes were over-transcribed in Martinique populations . The genulations . As in lCCEae3A, CYP6M11, CYP9M9. Larval resistance to temephos was negatively correlated to adult deltamethrin resistance. Adult deltamethrin resistance was mainly represented by the second PCA axis and positively correlated to urbanization, deltamethrin thermal fogging application and the adult over-transcription of CYP9M9, GSTE7 but negatively correlated to agriculture, sugar cane culture and the adult over-transcription of CYP6Z6 and CCEae3A. Deltamethrin application was positively correlated with Kdr mutation frequency. Variables related to agriculture were mainly represented by the second and third PCA axes and positively correlated together. Sugar cane culture and in a lesser extent other agriculture variables were positively correlated to temephos resistance but not to deltamethrin resistance. Finally, one should note that several groups of genes showed a strong correlation of their transcription levels across the three first PCA axes such as CYP6Z6, CYP6Z8, GSTE7 and GSTE2 in larvae; CCEae3A, CYP6M11 and CYP9M9 in larvae; CPR and CYP9J22 in larvae or CYP9M9 and GSTE7 in adults.Principal Component Analysis (PCAs) was performed on all Martinique populations with 29 variables including insecticide resistance levels, larval and adult transcription ratios of candidate genes and environmental variables. Relations among variables across all Martinique populations are presented in Ae. aegypti populations of Martinique were resistant to temephos and deltamethrin but mostly susceptible to Bti. For Bti, although a slight resistance to Bti toxins could not be excluded Bti remains the main insecticide available for larval treatments, the low level of resistance is encouraging for future vector control in Martinique.Toxicological results showed that all Kdr mutation was found in Martinique populations, confirming the results previously obtained by Saavedra-Rodriguez et al. Kdr mutation was observed among the nine populations, indicating that deltamethrin resistance is partly associated with target site mutation. Donnelly et al. Kdr genotype and susceptibility to DDT and pyrethroids in many mosquito species, including Ae. aegypti. Saavedra-Rodriguez et al. Kdr mutation regarding knock-down time, recovery and survival rate of Ae. aegypti adults exposed to pyrethroids. Our study revealed a wide range of survival rates after deltamethrin exposure (20% to 90%) while the V1016I Kdr mutation was almost fixed in all sampled populations (f≥0.87), suggesting that other resistance mechanisms may involved in mosquito test population from Martinique. One should note that two novel mutations in the sodium channel gene (mutation F1552C and F1534C) linked to pyrethroid and DDT resistance has been recently found in Ae. aegypti in Thailand Among target-site mutations conferring resistance to chemical insecticides, only the V1016I Ace.1 gene in this population should confirm the absence of this mutation in Martinique. Unless other mutations are present elsewhere in this gene, our results showed that organophosphate resistance is mainly due to metabolic mechanisms.Enzymatic phenotyping of AChE1 did not allow demonstrating the presence of the G119S and F290V mutations in organophosphate resistance in mosquito test population from Martinique. However, the RSAL population presented a slightly lower inhibition rates for dichlorvos and propoxur compared to other populations. The sequencing of the Ae. aegypti. Biochemical assays on adults showed that deltamethrin resistance seemed to be associated with higher P450 levels confirming previous results obtained by Marcombe et al. Ae. aegypti GST has yet been shown to metabolize pyrethroids or their metabolites. Four populations presented higher activities of α-CCEs (Ae. aegypti larvae from Martinique.By quantitative trait loci (QTL) mapping, Saavedra-Rodriguez et al. f α-CCEs  which haCYP6Z6, CYP6Z8, GSTE7 and CPR seemed to be over-transcribed to a similar extent at both life stages, while others showed a more pronounced over-transcription in adults (CYP9J22 and CCEae3A) or larvae (GSTE2). Such life-stage specific over-transcription patterns suggest that particular enzymes might be more specifically involved in resistance to chemical insecticides during a particular life stage At the molecular level, metabolic resistance of Martinique populations through over-expression of detoxification enzymes was investigated by quantitative RT-PCR on 12 candidate genes. Our results showed that several candidate genes were over-transcribed in Martinique populations comparatively to the susceptible strains. Among them, CYP6Z subfamily has been previously associated with response to pyrethroid, carbamate and organochlorine insecticides Ae. aegypti, CYP6Z9 has been found 4-fold over-transcribed in a permethrin-resistant mosquito population collected in Northern Thailand CYP6Z8 was also identified as inducible by permethrin and other pollutants CYP6Z6 and CYP6Z8 in most Martinique populations confirms the possible involvement of Ae. aegypti CYP6Zs in insecticide resistance in Martinique. The P450 gene CYP9M9 was found over-transcribed in several Martinique populations at both life stages. This gene was found to be inducible by permethrin, temephos and others pollutants CYP9M9 was also over-transcribed in larvae of the susceptible strain SBE from Benin and showed important variations between populations, suggesting that this gene may not have a major role in resistance. Conversely, the repeated over-transcription of CYP9J22 in adults is in agreement with results obtained by Marcombe et al. Ae. aegypti CYP9Js to metabolize pyrethroids was validated by heterologous expression followed by in-vitro insecticide metabolism assays , confirming the involvement of this P450 subfamily in insecticide resistance. Finally, Lycett et al.CPR) causes an increased susceptibility to permethrin in An. gambiae. Therefore, the recurrent elevated transcription level of the CPR gene at both life stages supports the major role of the P450 detoxification system in metabolic resistance mechanisms in Martinique.Over-transcription of genes encoding P450s has been frequently associated with metabolic-based insecticide resistance in insects GSTE2 is of particular interest since the associated enzyme has been shown to metabolize DDT in An. gambiae and Ae. aegyptiGSTE2 was also found over-transcribed in a DDT- and pyrethroid-resistant mosquito population from Thailand GSTE2 in Martinique. The gene GSTE7 was also found over-transcribed in several Martinique populations, and in several pyrethroid resistant Ae. aegypti strain from Thailand The over-transcription of the gene CCEae3A was constitutively over-transcribed in all Martinique populations at both life stages and highly correlated with temephos resistance. The massive use of temephos during decades in Martinique may have selected for a CCE based metabolic resistance and particularly for the CCEae3A gene. Further work on the phenotypic expression of this candidate gene in Aedes would be required as esterases are known to play an important role in OPs resistance in mosquitoes The esterase gene Kdr mutation frequency possibly associated to the selection pressure applied in urban zones by deltamethrin treatments. Recent studies  showed a surprising strong genetic differentiation of the Aedes populations of Martinique. Larval or adult treatments especially in urban zone may have an influence on the resistance selection, the effective population size (bottleneck) and on the population structure. However, the correlation between Kdr mutation frequency and deltamethrin resistance was not so significant, confirming that metabolic resistance mechanisms play a significant role in deltamethrin resistance and that other sources of selection exist like household insecticides. The agriculture and sugar cane culture variables were correlated with larval resistance to temephos suggesting a potential role of agriculture in the selection of organophosphate resistance in Martinique. Many studies showed that agricultural practices such as cotton or vegetable culture may have an essential role in the selection for DDT and pyrethroid resistance, especially for the main malaria vector Anopheles gambiae in Africa Ae. aegypti is mainly associated to urban and peri-urban environments explain the lesser impact of agricultural practices on this resistance pattern.The principal component analysis of 29 variables characterizing the 9 populations of Martinique underlined potential relationships between variables. Negative correlation between resistance to temephos and to deltamethrin suggests not only the absence of cross-resistance between the two insecticides but also that both resistances are submitted to different selection pressures in Martinique. The absence of correlation between deltamethrin resistance and agriculture variables may indicate that agricultural pesticides do not play a major role on adult deltamethrin resistance in Martinique or eventually that this impact is too homogenous across populations to be highlighted by our analysis. A significant relationship appeared between urbanization, deltamethrin application and Kdr mutation and the over-production of detoxification enzymes such as with CYP6Zs and GSTs participating in phase I and phase II detoxification steps respectively. At the larval stage an interesting positive correlation was observed between CCEae3A transcription level and temephos resistance, confirming the potential role of this gene in metabolic resistance of this insecticide. However, such correlation was also observed with the P450 genes CYP6M11 and CYP9M9 suggesting that multiple detoxification enzyme families may be involved in temephos resistance.From the present study, it could be hypothesized that deltamethrin resistance in Martinique results from the combination of the presence of the V1016I Ae. aegypti Martinique populations.This study showed that resistance to chemical insecticides is multiple and widely distributed among CYP6Z, CYP9J or CYP6M subfamilies, epsilon GSTs or the esterase CCEae3A represent good candidates for further functional validation. Although it cannot be excluded that other genes are involved in insecticide resistance, gene silencing and heterologous expression approaches will provide more evidences of their potential role in metabolic resistance.Several detoxification genes such as P450s belonging to the Ae. aegypti resistance to insecticide in Martinique was preliminarily investigated through a multivariate analysis. Additional population genetic and genomic studies are currently performed on Ae. aegypti Martinique populations. These studies should bring more information about the genetic structure of Martinique populations and eventually point out specific genetic region under selection. A better understanding of the genetic basis of insecticide resistance is essential to optimize vector control strategies.The role of environmental factors such as agriculture in Information S1Variables used for PCA analyses.(XLS)Click here for additional data file.Information S2Detailed PCA analysis.(XLS)Click here for additional data file.


Inclusion or exclusion of single codons at the splice acceptor site of mammalian genes is regulated in a tissue-specific manner, is strongly conserved, and is associated with local accelerated protein evolution. Drosophila. Alternative splicing of NAGNAGs in human was associated with dramatically increased frequency of exon length changes at orthologous exon boundaries in rodents, and a model involving point mutations that create, destroy, or alter NAGNAGs can explain both the increased frequency and biased codon composition of gained/lost sequence observed at the beginnings of exons. This study shows that NAGNAG alternative splicing generates widespread differences between the proteomes of mammalian tissues, and suggests that the evolutionary trajectories of mammalian proteins are strongly biased by the locations and phases of the introns that interrupt coding sequences.Thousands of human genes contain introns ending in NAGNAG (N any nucleotide), where both NAGs can function as 3′ splice sites, yielding isoforms that differ by inclusion/exclusion of three bases. However, few models exist for how such splicing might be regulated, and some studies have concluded that NAGNAG splicing is purely stochastic and nonfunctional. Here, we used deep RNA-Seq data from 16 human and eight mouse tissues to analyze the regulation and evolution of NAGNAG splicing. Using both biological and technical replicates to estimate false discovery rates, we estimate that at least 25% of alternatively spliced NAGNAGs undergo tissue-specific regulation in mammals, and alternative splicing of strongly tissue-specific NAGNAGs was 10 times as likely to be conserved between species as was splicing of non-tissue-specific events, implying selective maintenance. Preferential use of the distal NAG was associated with distinct sequence features, including a more distal location of the branch point and presence of a pyrimidine immediately before the first NAG, and alteration of these features in a splicing reporter shifted splicing away from the distal site. Strikingly, alignments of orthologous exons revealed a ∼15-fold increase in the frequency of three base pair gaps at 3′ splice sites relative to nearby exon positions in both mammals and in  In order to translate a gene into protein, all of the non-coding regions (introns) need to be removed from the transcript and the coding regions (exons) stitched back together to make an mRNA. Most human genes are alternatively spliced, allowing the selection of different combinations of exons to produce multiple distinct mRNAs and proteins. Many types of alternative splicing are known to play crucial roles in biological processes including cell fate determination, tumor metabolism, and apoptosis. In this study, we investigated a form of alternative splicing in which competing adjacent 3′ splice sites (or splice acceptor sites) generate mRNAs differing by just an RNA triplet, the size of a single codon. This mode of alternative splicing, known as NAGNAG splicing, affects thousands of human genes and has been known for a decade, but its potential regulation, physiological importance, and conservation across species have been disputed. Using high-throughput sequencing of cDNA (“RNA-Seq”) from human and mouse tissues, we found that single-codon splicing often shows strong tissue specificity. Regulated NAGNAG alternative splice sites are selectively conserved between human and mouse genes, suggesting that they are important for organismal fitness. We identified features of the competing splice sites that influence NAGNAG splicing, and validated their effects in cultured cells. Furthermore, we found that this mode of splicing is associated with accelerated and highly biased protein evolution at exon boundaries. Taken together, our analyses demonstrate that the inclusion or exclusion of RNA triplets at exon boundaries can be effectively regulated by the splicing machinery, and highlight an unexpected connection between RNA processing and protein evolution. The split structure of eukaryotic genes impacts gene expression and evolution in diverse ways. Most directly, the presence of introns enables multiple distinct mRNA and protein products to be produced from the same gene locus through alternative splicing, which is often regulated between tissues or developmental stages Alternative 3′ and 5′ splice site use, in which longer or shorter versions of an exon are included in the mRNA, are among the most common types of alternative splicing in mammals NAGNAG alternative splicing  has beenDrosophila. NAGNAG isoforms can be uniquely distinguished by short reads that overlap the splice junction, and the quantity of data available from each tissue in human and mouse typically represented at least 80-fold mean coverage of the transcriptome, a depth sufficient to detect potential tissue-specific differences in many cases. Sequence features were identified which can shift splicing toward the proximal or distal NAG, providing clues to regulation. We also analyzed the impact of NAGNAGs on exon evolution, obtaining evidence that NAGNAGs dramatically accelerate addition and deletion of sequence at the beginnings of exons.In order to assess the abundance and potential regulation of NAGNAG splicing events genome-wide, we analyzed polyA-selected RNA-Seq data generated using the Illumina HiSeq platform from 16 human tissues at depths of ∼8 Gbp per tissue, similarly deep RNA-Seq data that we generated from eight mouse tissues, and data generated by the modENCODE consortium across a developmental time course in Our initial analyses used the Illumina Body Map 2.0 dataset of polyA-selected RNA-Seq data from 16 human tissues  sequenced at depths of ∼80 million paired-end 2×50 bp reads per tissue. This sequencing depth generates ∼8 Gbp of data, representing >80-fold coverage of the human protein-coding transcriptome. Enumerating all possible NAGNAG splicing events, we mapped both ends of each read against NAGNAG splice junctions . Isoformp<0.01 by multinomial test). Furthermore, approximately 42% were “strongly regulated,” with changes in ψ of at least 25% between tissues  using RNA-Seq data from the modENCODE consortium Drosophila genes where both isoforms were expressed at ≥5% in at least one developmental time point. Of these, 14% were developmentally regulated, with 5% being strongly regulated as defined above. As in mammals, more highly regulated fly NAGNAGs were associated with increased sequence conservation within and upstream of the 3′ splice site  predict predominant proximal splice site usage, since C is strongly favored over A and is also proximal, but roughly equal proportions of both isoforms are expressed across all tissues studied  nucleotides at both −3 bases (YAGYAGs). We found that exons that predominantly used the proximal splice site  had substantially distinct nucleotide preferences from those that predominantly used the distal site  , consistStrongly regulated YAGYAGs had features that were intermediate between the extremes found for proximal major and distal major YAGYAGs, such as polypyrimidine tracts of intermediate length , suggestThe −4 base, four nucleotides upstream of the 3′ splice site, is not generally considered to be important in splicing  was intact or in a context in which the native BPS had been replaced by the previously mapped BPS of IGF2BP1 intron 11 . With thTogether, our analyses of proximal/distal major splicing suggested that NAGNAG 3′ splice sites afford broad scope for evolutionary tuning of isoform ratios, even in cases where the sequence of the second NAG is constrained by selection on the encoded amino acid. For example, mutations affecting the upstream −3 and −4 bases, the polypyrimidine tract, or the location of the branch site could all potentially modulate the ratio of the two isoforms across a range from predominantly proximal to predominantly distal isoform usage, which might facilitate evolutionary addition and deletion of single codons at 3′ splice junctions. A previous study observed reduced frequencies of amino acid substitutions near exon-exon junctions relative to the centers of exons, presumably resulting from purifying selection acting on exonic splicing enhancer motifs While gain/loss of exonic sequence is normally attributed to insertions or deletions (“indels”) in the genome, the increased frequency of changes at the 3′ splice site suggested a prominent role for an alternative mechanism involving genomic substitutions that give rise to three base shifts in exon boundaries without insertion or deletion of genomic DNA. For example, creation of a NAG motif immediately upstream of a 3′ splice site NAG by mutation would be expected to commonly shift splicing upstream by three bases (resulting in exonization of three bases of intron) or generate an alternatively spliced NAGNAG that could subsequently lose splicing at the downstream NAG through mutation. Alternatively, a mutation creating an immediately downstream NAG—or a mutation that weakened the upstream NAG relative to a pre-existing downstream NAG—could result in either alternative splicing or loss of three bases of exonic sequence. As outlined in Drosophila lineage, we observed similar enrichment for gain/loss of three bases of exonic sequence at the 3′ splice site when comparing orthologous D. melanogaster and D. yakuba coding exons  for only 18% of NAGNAGs with favorable pyrimidine bases at both −3 positions based on RNA-Seq read depths slightly below those used in human. By contrast, 50%–85% of human, mouse, and Drosophila YAGYAGs were detected as alternatively spliced, suggesting that NAGNAG alternative splicing is substantially rarer in worms than in other metazoans. This decrease in abundance mirrors the 3-fold weaker enrichment of three base gaps at 3′ splice sites observed in worms , we observed evidence of residual NAGNAG motifs in exons with altered boundaries . These data are also accessible from ArrayExpress (ArrayExpress accession: E-MTAB-513). The Body Map 2.0 data were generated by the Expression Applications R&D group at Illumina using the standard (polyA-selected) Illumina RNA-Seq protocol from total RNA obtained commercially (Ambion) using the HiSeq 2000 system. We downloaded D. melanogaster  and C. elegans  RNA-Seq data from the modMINE (http://intermine.modencode.org/) website of the modENCODE consortium. For the C. elegans data, we restricted to 36 bp reads for consistency with other analyses.Mapped sequence reads from the human and mouse RNA-Seq experiments are located in NCBI's GEO database (accession number GSE30017). The complete Body Map 2.0 sequence data are in the ENA archive with accession number ERP000546 . Tissue was lysed in Trizol and RNA was extracted with Qiagen miRNeasy mini columns. Using 5 µg of total RNA, we performed polyA selection and prepared strand-specific libraries for Illumina sequencing following the strand-specific dUTP protocol p≤0.01 by the proportion or z-test (prop.test in R [http://www.R-project.org/]). As described in the text, when computing the fraction of regulated NAGNAGs, we only considered NAGNAGs which were alternative spliced by these criteria (both isoforms expressed at ≥5% in at least one sample).For each NAGNAG, we extracted the sequence flanking the proximal and distal 3′ splice sites and used Bowtie For p≤0.01 between the replicates. We estimated mean FDRs for human (4.4%) and mouse (1.1%) by taking a weighted average over tissues, where we weighted the FDR computed for each tissue by the number of alternatively spliced NAGNAGs analyzed for that tissue.We estimated false-discovery rates as the fraction of events which were differentially expressed between technical (human) or biological (mouse) replicates identified using the procedure described above for regulated events. Briefly, for each tissue and pair of replicates, we restricted to the set of NAGNAGs which were alternatively spliced in at least one of the replicates and computed the fraction of these NAGNAGs which were differentially expressed with The fraction of strongly regulated NAGNAGs increased essentially linearly with the number of tissues considered for both human and mouse . We expeFor each NAGNAG event, the probabilities of using the proximal and distal splice sites are proportional to http://www.biostat.wisc.edu/~cdewey/mercator/) and FSA Drosophila analysis, we used a previously described D. melanogaster–D. yakuba whole-genome alignment We performed a whole-genome alignment of human and mouse using Mercator . The proximal (63 nucleotides) and distal (60 nucleotides) isoforms were resolved on a 10% TBE gel and detected with a Typhoon 9000 scanner . Proximal and distal isoforms were quantified with ImageJ software.All minigene reporters (0.5 µg) were transfected into HEK293T cells using Lipofectamine 2000 (Invitrogen). RNA was isolated 18–24 h post-transfection with RNeasy Mini Kits (Qiagen). RT-PCR was performed with a fluorescent primer . For each exon in each class, we extracted the corresponding intronic sequence and created a sequence logo using WebLogo .Unless otherwise described, all plots in Figure S1Dependence of the fraction of strongly regulated NAGNAGs on the number of tissues. (A) Human. (B) Mouse.(TIFF)Click here for additional data file.Figure S2Technical variability in human libraries. Single-end (75 bp) and paired-end (2×50 bp) sequencing of the same human libraries captures sequencing variability.(TIFF)Click here for additional data file.Figure S3Biological variability in mouse libraries. Sequencing of mouse libraries created from two different individuals captures all major sources of variability, including library preparation (2×36 bp versus 2×80 bp), sequencing, sample collection, and individual-specific splicing (C57BL/6J versus DBA/2J).(TIFF)Click here for additional data file.Figure S4Correlation between human and mouse isoform usage patterns for NAGNAGs with diverged differences in splice site scores. As (TIFF)Click here for additional data file.Figure S5Biophysical models of NAGNAG isoform usage in different species. (A) Human Click here for additional data file.Figure S6Isoform usage of the NAGNAG in the PTBP2 gene illustrated in (TIFF)Click here for additional data file.Figure S7Splice site score difference and maximum splice site score as a function of switch score for different classes of alternative 3′ splice sites. (A) The splice site scores of regulated NAGNAG 3′ splice sites tended to be far more similar to one another than those of unregulated events, suggesting that regulation is easier to achieve when the intrinsic strengths of the sites are evenly matched. (B–C) This trend was much weaker for more distant alternative 3′ splice site events. (D) The 3′ splice site scores of tissue-regulated NAGNAGs also tended to be somewhat weaker than for unregulated NAGNAGs or constitutive 3′ splice sites. This observation suggested that weaker splice sites are more easily regulated, consistent with previous studies of other types of alternative splicing. (E–F) This trend for regulated events to be associated with weaker splice site scores was observed to a much lesser extent for alternative 3′ splice sites separated by longer distances, suggesting that splicing regulatory elements may more readily exert differential effects on more widely spaced 3′ splice sites, making matching of splice site scores less critical for achieving regulation for this class than it is for NAGNAGs. For example, we have previously shown that most exonic splicing silencer (ESS) elements inhibit the intron-proximal site when situated between competing 3′ splice sites, an arrangement that requires separation of the competing sites by sufficient space to accommodate the ESS, and so does not apply to NAGNAGs. “v. low” indicates “very low,” and “CJ” indicates the 3′ splice sites of constitutive junctions.(TIFF)Click here for additional data file.Figure S8Relative conservation at the −4 position for different classes of NAGNAGs. Plot shows median relative conservation at the −4 position, computed as (phastCons score at −4 position/phastCons score at −3 position). “CJ” indicates the 3′ splice sites of constitutive junctions. Error bars indicate the standard error of the median, estimated by bootstrapping.(TIFF)Click here for additional data file.Figure S9Numbering of alignment gaps relative to the 5′ and 3′ splice sites. Examples shown in the figure illustrate the numbering system used for assessing gap positions relative to the 5′ and 3′ splice sites. The splice sites are numbered 0, and gap position is numbered relative to the nearest splice site. Gaps that could not be unambiguously assigned to one splice site were very rare and their inclusion or exclusion did not affect our conclusions.(TIFF)Click here for additional data file.Figure S10Exons with constitutively spliced NAGNAGs show an enrichment for gaps at the 3′ splice site. We restricted our analysis in (TIFF)Click here for additional data file.Figure S11Alignment gaps at splice sites are enriched for predicted phosphorylation sites. The distribution of alignment gaps containing one or more predicted phosphorylation sites is shown for (A) all gaps and (B) gaps of three bases.(TIFF)Click here for additional data file.Table S1Abundance and regulation of alternative splicing events in human protein-coding sequence (paired-end sequencing). “No. of events” shows the abundance of alternative splicing events in protein-coding sequence, restricted to events for which (1) neither isoform is predicted to be targeted by nonsense-mediated decay (no splice junction ≥50 nt downstream of the stop codon), and (2) both isoforms are expressed at ≥5% in at least one tissue. Isoform ratios are based on the numbers of reads aligning to the 3′ splice sites of each isoform, thereby treating each event as a choice between competing 3′ splice sites . This method ensures that the different classes of splicing events are analyzed “fairly,” irrespective of the length of the alternatively spliced sequence. “Fraction strongly regulated” gives raw estimates .(DOCX)Click here for additional data file.Table S2Complete list of human genes containing alternatively spliced NAGNAGs, sorted by estimated switch score.(XLS)Click here for additional data file.Table S3Abundance and regulation of alternative splicing events in human protein-coding sequence (single-end sequencing). Identical to (DOCX)Click here for additional data file.Table S4Abundance and regulation of alternative splicing events in mouse protein-coding sequence. Similar to (DOCX)Click here for additional data file.Table S5Complete list of mouse genes containing alternatively spliced NAGNAGs, sorted by estimated switch score.(XLS)Click here for additional data file.Table S6Models for evolutionary gain and loss of single codons at 3′ splice sites resulting from splicing changes caused by single nucleotide substitutions. For each major category of change (bold headings in first column), the second column diagrams the effect on splicing, e.g., /… ⇒ …/ indicates a three base shift downstream in the location of the 3′ splice site, and …/ ⇒ /…/ indicates a change from constitutive splicing to alternative splicing at both the original 3′ splice site and a site three bases upstream. Below each diagram is a sequence motif consisting of specific bases , degenerate positions , or partially degenerate positions . The third column lists conditions that are expected to favor each type of change (based on −3 base preferences shown in (DOCX)Click here for additional data file.Table S7Number of amino acids gained or lost through 3′ splice site gaps are strongly biased depending on the intron phase. Table shows counts of amino acids gained/lost between orthologous human and mouse exons; the corresponding frequencies are shown in (DOCX)Click here for additional data file.


Dynamin 1–3 isoforms are known to be involved in endocytotic processes occurring during synaptic transmission. No data has directly linked dynamins yet with normal animal behavior. Here we show that dynamin pharmacologic inhibition markedly impairs hippocampal-dependent associative memory. Memory loss was associated with changes in synaptic function occurring during repetitive stimulation that is thought to be linked with memory induction. Synaptic fatigue was accentuated by dynamin inhibition. Moreover, dynamin inhibition markedly reduced long-term potentiation, post-tetanic potentiation, and neurotransmitter released during repetitive stimulation. Most importantly, the effect of dynamin inhibition onto memory and synaptic plasticity was due to a specific involvement of the dynamin 1 isoform, as demonstrated through a genetic approach with siRNA against this isoform to temporally block it. Taken together, these findings identify dynamin 1 as a key protein for modulation of memory and release evoked by repetitive activity. All efforts were made to minimize the number of animals used and their suffering. Animals were housed 4–5 per cage after weaning.All animals (C57BL/6J mice) were used and handled in strict accordance with good animal practice as defined by the Ethical Guidelines for Treatment of Laboratory Animals of Columbia University and specifically approved by Columbia University IACUC (protocol #AC-AAAB8674). Specific Pathogen Free, male mice, aged 4–5 months, were obtained from Jackson Laboratories. Mice were housed under a 12-h (8.00–20.00) light/dark cycle in a climate-controlled room (23°C±1) with 2HPO4 1 mM, NaHCO3 25 mM, Glucose 10 mM, CaCl2 2 mM, MgCl2 2 mM, supplemented with 0.3% DMSO. Drug solution was then either perfused for 20 minutes onto hippocampal slice preparations or injected into dorsal hippocampi.Dynasore, kindly provided by Tomas Kirchhausen  was dissolved in anhydrous DMSO to obtain a 200 mM stock concentration and then stored at −80°C. Working solutions were obtained by diluting, in dim light environment, aliquots of stock solutions in artificial cerebro-spinal fluid (ACSF) containing NaCl 124 mM, KCl 4.4 mM, Na5′(S-S) UAA GUG UCA AUC UGG UCU C dTdT 3′” while the siRNA sequence for the control siRNA was “5′(S-S) CGU ACG CGG AAU ACU UCG AUU dTdT 3′”. Annealed siRNA duplexes were re-suspended in K+ buffer, incubated at 90°C and then treated with an equimolar mixture of TCEP , a strong reducing agent, at 20°C for 15 min. An equimolar ratio of monomeric Penetratin1  was then incubated at 65°C for 15 min with the siRNA mixture, followed by a further incubation at 37°C for 1 hr, as previously described siRNA duplexes with a 5′ thiol on the sense strand were synthesized . The siRNA sequence against Mus musculus dynamin 1 was: “Bilateral cannulas were fixed to the skull, as previously described All behavioral studies were performed by an observer who did not know the treatment group of the mice until the entire test had been completed, and conducted during the light phase (between 13:30–18:00) of the light/dark cycle (lights on 8:00–20:00).Contextual and cued learning were assessed using the fear conditioning test, as previously described 3–CA1 field excitatory post-synaptic potentials (fEPSP) were recorded by placing the stimulating electrode on CA3 fibers projecting to CA1stratum radiatum where the recording electrode was placed fEPSPs to generate input-output relations before each experiment. For the long-term potentiation (LTP) experiments, a 30-minute baseline was recorded every minute at an intensity that evokes a response approximately 35–40% of the maximum evoked response. In most of the experiments LTP was induced using a theta-burst stimulation . In a few experiments, LTP was induced through a different protocol  that is known to solely involve post-synaptic mechanisms A  and GABAB  receptor blockers, or in the presence of an AMPA receptor desensitization blocker . For both PTP measurement and SF assessment, the tetanic stimulation was applied in the constant presence of 100 μM D-APV, a competitive NMDA receptor antagonist. PPF analysis was carried out by providing a double stimulation delivered at different interstimulus intervals (10→1000 ms) and then comparing the ratio between the slope of the evoked response after the second stimulus (S2) vs. the slope of the evoked response obtained for the first stimulus (S1). Tetanic efficiency as an evaluation of the facilitation induced throughout the tetanus (an indirect index of neurotransmitter release) was evaluated through intra-burst analysis Mice were sacrified by cervical dislocation followed by decapitation. Transverse hippocampal slices (400 μm) were obtained by using a tissue-chopper and maintained in an interface chamber at 29°C. CAAll data are shown as mean ± SEM. Statistical tests included two-way ANOVA with repeated measures for multiple comparisons or two-tails Mann-Whitney test when appropriate.In a first series of experiments, we checked whether memory is affected by dynasore, a synthetic non-peptidic inhibitor of dynamins d-aspartate (NMDA) receptor blocker D-aminophosphovaleric acid (D-APV) (100 μM), leaving intact 2-amino-3--propanoic acid (AMPA) receptor-mediated events A receptor antagonist, and SCH 50911 (100 μM), a GABAB receptor antagonist, applied to antagonize the GABAergic input, was not capable of protecting from the further decay rate induced by dynasore  is an activity-dependent phenomenon widely proposed as a cellular model for learning and memory before  - but nobefore  a theta-fEPSP input/output relationships. These results are consistent with studies showing that basal evoked exocytosis is not affected by dynamin inhibition in rodent neurons Changes in evoked responses during sustained activity were not associated, in turn, with significant modification of basal neurotransmission . TreatmeThe theta-burst stimulation protocol used in the experiments reported above is known to produce a compound LTP involving both pre- and post-synaptic mechanisms during the tetanus reduction of presynaptic input along the tetanus. Interestingly, the reduction in area was present within the first burst of 4 pulses at 100 Hz . This indicates that the efficacy of the theta-burst stimulation in triggering the compound LTP is not conserved upon dynamin inhibition, and supports the possibility that dynamin block reduces presynaptic input.To test whether a possible decrease of neurotransmitter vesicle availability produced by dynamin inhibition would decrease tetanic stimulation efficacy in inducing plasticity, we measured the depolarization occurring t 100 Hz  and beca2+ elevation within the presynaptic terminal during high frequency stimulation An additional evidence in favor of a dynamin inhibition-induced impairment of neurotransmitter released during sustained activity derived from studies on post-tetanic potentiation (PTP). This is a type of short-term synaptic plasticity dependent upon Ca2+ concentration for a very short time Paired-pulse facilitation (PPF) is a short-term plasticity phenomenon that, differently than SF, PTP and LTP, is not due to sustained release. It occurs when a synaptic response is enhanced by a preceding stimulation of similar intensity, presumably elevating the CaDynamin has three different isoforms, dynamin 1–3. Given that dynasore is not selective toward a specific dynamin isoform The main finding of the present work is that dynamin 1 is important for the formation of associative memory in hippocampus. An additional finding is that both short- and long-term plasticity phenomena caused by fast and prolonged activity and thought to be linked to memory formation, depend upon dynamin function. Specifically, we have demonstrated that SF, LTP, PTP, and tetanus efficacy, all phenomena due to sustained release, are markedly reduced following dynamin inhibition.ts1shibire, a temperature-sensitive dynamin mutant gene in Drosophila melanogaster, alters neuronal mechanisms that are thought to be linked to learning and memory Lymnaea stagnalis is associated with increased expression of syntaxin 1 and dynamin 1 coincident with elevated CREB 1 levels, and knockdown of CREB1 prevented memory consolidation and reduced expression of both syntaxin and dynamin 1 Btbd9 knock-out mice displayed an increase in dynamin 1 expression together with enhanced synaptic plasticity and memory The involvement of dynamin 1 in memory function opens new perspectives to studies on the function of the protein. Specifically, it translates effects of inhibition of this isoform into animal behavior, and memory in particular. These data are consistent with the observation that a priori, given that other proteins are still able to increase memory and synaptic plasticity while decreasing the presynaptic input The role of different forms of plasticity involving dynamin in evoked release during sustained activity is also very interesting. It is noteworthy to remark the fact that memory formation is not always linearly paired with the level of neurotransmitter release as one might assume 3–CA1 connection is not a single-compartment phenomenon, but rather a mix of both presynaptic and postsynaptic processes that independently strengthen the synapse. Theta-burst CA3-CA1 LTP activates second messanger cascades including the cAMP-PKA pathway either in the presynaptic terminal to directly boost presynaptic function or in the postsynaptic site to produce a retrograde messenger as well as activate glutamate receptors and L-voltage-gated calcium channels in the post-synaptic neuron LTP at CAafter the tetanic stimulation suggesting, in turn, that dynamin inhibition affects memory acquisition. Dynamin activity becomes essential for memory and plasticity induced by high frequency stimulations, such as theta burst stimulation, in line with the independent findings of Ferguson et al de facto the vesicle availability for prompt release. This suggests that, at least at high frequency stimulation, even the slow vesicle recovery due to the classic clathrin-dependent/dynamin-dependent endocytosis is necessary to compensate the intense vesicle use for rapid release due to tetanic stimulation. Our data on contextual fear conditioning and LTP are novel because they correlate the possible efficiency of dynamin-dependent vesicle availability selectively with memory and plasticity phenomena, while our data on basal synaptic transmission confirm that synaptic potentials not evoked by sustained release are not affected by residual vesicle availability upon dynamin inhibition. Further investigations, beyond the scope of this work, are required to discern whether solely the classical or any alternative endocytotic pathways or even non endocytosis-related mechanisms are critical for dynamin-dependent memory and synaptic plasticity.Recent evaluations of vesicle dynamics In our studies, we provide both pharmacologic and genetic proof of the involvement of dynamin in memory formation. There are several advantages with using a genetic approach in addition to a pharmacologic one. The experiments with dynamin 1 siRNA provide an independent genetic validation of the findings with dynasore. Furthermore, they demonstrate that the isoform 1 of dynamin is active in memory and synaptic plasticity. Finally, they offer precious insight into the complementarity of the three major dynamin isoforms. Neither dynamin 2 nor dynamin 3 were capable of compensating for the detrimental effect exerted on cognition and plasticity by selective dynamin 1 knockdown obtained through siRNA technique. Similarly, the presence of dynamin 2 and 3 was not able to avoid death in dynamin 1-knockout mice at two weeks of age Genetic ablation of dynamin 1 produced a less marked effect onto synaptic plasticity than the pharmacologic block with dynasore. Possible explanations for the incomplete block of LTP by the siRNA are likely to be linked to the fact that dynasore inhibits multiple dynamin isoforms whereas the siRNA blocks selectively dynamin 1, and/or that dynasore affects substrates other than dynamins de facto the number of vesicle recovered and ready to be reused) does not mimic the clear stimulation-frequency-dependent release depression after treatment with dynasore or a clathrin inhibitor treatment, while it efficiently blocks vesicular refilling As previously reported, the level of neurotransmitter release during ongoing activity may be regulated by the availability of release-ready vesicles and, therefore, depends on the speed of vesicle recruitment. In that sense, synaptic vesicles need to undergo fast recycling to prevent depletion of the synaptic vesicle pool


A combination consisting of gefitinib and sunitinib acted synergistically in inhibiting growth of GBM oncospheres in vitro. Sunitinib was the only RTK inhibitor that could induce apoptosis in GBM cells. However, the in vivo efficacy testing of the gefitinib and sunitinib combination in an EGFR amplified/ PTEN wild type GBM xenograft model revealed that gefitinib alone could significantly improve survival in animals whereas sunitinib did not show any survival benefit. Subsequent testing of the same drug combination in a different syngeneic glioma model that lacked EGFR amplification but was more susceptible to sunitinib in vitro demonstrated no survival benefit when treated with gefitinib or sunitinib or the gefitinib and sunitinib combination. Although a modest survival benefit was obtained in one of two animal models with EGFR amplification due to gefitinib alone, the addition of sunitinib, to test our best in vitro combination therapy, did not translate to any additional in vivo benefit. Improved targeted therapies, with drug properties favorable to intracranial tumors, are likely required to form effective drug combinations for GBM.Glioblastoma multiforme (GBM) is the most common intracranial cancer but despite recent advances in therapy the overall survival remains about 20 months. Whole genome exon sequencing studies implicate mutations in the receptor tyrosine kinase pathways (RTK) for driving tumor growth in over 80% of GBMs. In spite of various RTKs being mutated or altered in the majority of GBMs, clinical studies have not been able to demonstrate efficacy of molecular targeted therapies using tyrosine kinase inhibitors in GBMs. Activation of multiple downstream signaling pathways has been implicated as a possible means by which inhibition of a single RTK has been ineffective in GBM. In this study, we sought a combination of approved drugs that would inhibit  Improving therapy for patients with Glioblastoma multiforme (GBM) is one of the biggest challenges in oncology. Although molecular targeting has shown success in many cancers, targeted therapy for GBM has yet to demonstrate an appreciable clinical survival benefit The oncogenic receptor tyrosine kinases (RTKs) that are mutated in GBM are obvious molecular targets and many small molecule inhibitors of the RTKs are available. A mutation analysis of over 20,000 gene coding regions in GBM genomes confirmed that the RTK/PI3K/AKT pathway is one of the most frequently altered groups of genes in GBM Lack of successful therapies against GBMs using RTK inhibitors raises several questions. Are the molecular targeting agents reaching and inhibiting the presumed target effectively in GBM? What are the resistance mechanisms involved if the inhibitors are reaching the tumor in effective concentrations? Growth signaling through alternate pathways, as well as tumor heterogeneity could be two of many factors involved in tumor resistance mechanisms.in vitro and in vivo to determine if we could find a combination of RTK inhibitors that would be more successful than a single agent. The premise of the work was to evaluate approved inhibitors designed to target the most frequently activated tyrosine kinases in GBMs. The best in vitro pair of drugs inhibited GBM oncospheres synergistically was gefitinib and sunitinib. However, the improved activity of RTK combination did not perform as predicted in vivo. Gefitinib alone had a significant but modest survival benefit in a GBM xenograft mouse model mouse model. Moreover, in vivo evaluation of the same drugs in a syngeneic rat model of GBM failed to provide any survival benefit. Although the single agent therapy might show activity in certain genetic backgrounds, combinations that effectively target multiple RTK pathways in an intracranial target are needed.In the following study, we tried to evaluate a series of RTK inhibitors in GBM systems in vitro cell-based assays for detecting activity of RTK inhibitors and combinations of inhibitors. For this we deemed it important that the cell lines were: 1) from human GBM patients 2) had relevant RTK pathway mutations or activation and 3) formed invasive grade IV astrocytomas when injected intracranially in nude mice.Our first goal was to develop Therefore, we employed GBM oncospheres for determining the effects of the RTK inhibitors on proliferation and cell death. Oncospheres, also referred to as stem-like cell cultures, grow in suspension using serum-free stem cell media. This culturing system appears to maintain genomic and phenotypic changes of the primary tumor better than traditional cell lines We used two GBM oncosphere lines for screening drug combinations. The 020913 GBM cell line maintains the primary tumor EGFR amplification as determined by a genomic copy number analysis The 060919 GBM cell line was derived from a xenograft tumor that was sequenced as part of a GBM genome sequencing project To investigate the active cell signaling pathways in GBM stem-like cells, 020913 and 060919 cells were analyzed using the phospho-RTK array and phospho-kinase array. These arrays simultaneously determine relative phosphorylation levels in over 40 different kinases.Analysis of the subsequent phosphorylation profiles revealed that both the GBM oncosphere cell lines were associated with extensive activation of multiple tyrosine kinases including both receptor and non-receptor tyrosine kinases as shown their phosphorylation status . The co-These results indicate that multiple kinases are activated in GBM oncosphere cells and have been summarized in 50 values of these eleven inhibitors were evaluated using an alamarBlue based assay and are listed in Eleven different RTK inhibitors were evaluated for their ability to inhibit GBM oncosphere growth. The IC50 concentrations. Thirty-two such combinations were evaluated and showed that single agents, did not substantially alter growth, and only certain combinations suppressed growth . The extent of growth fold inhibition was calculated by dividing the alamarBlue fluorescence values for the treated cells with fluorescence values for cells treated with the vehicle.To initially test our hypothesis of combined inhibition, the various pair-wise combinations of RTK inhibitors were tested for growth suppression at 25% (one fourth) and 10%  of their respective IC50 values were next used. Single drugs and pair-wise combinations of these drugs were analyzed in GBM oncosphere lines for proliferation and caspase induction. Drug combinations containing sunitinib were best at inducing apoptosis  and one fourth the ICpoptosis , and thepoptosis .50 concentrations for 24 hours. The drugs were withdrawn after 24 hours and the ability of the oncospheres to regrow was assessed after two additional weeks of culture in the growth media using alamar blue cell growth assay. The growth assessment revealed that oncospheres treated with single agents or with the combination of RTK inhibitors were able to regrow with the exception of the cells treated with the combination of gefitinib and sunitinib , JAK-STAT, SRC, c-JUN and p53. There was an observed increase in phosphorylation of IGF-IR and EphA2 .in vitro RTK combination consisting of gefitinib and sunitinib was evaluated in vivo in an intracranial glioblastoma xenograft model. Five hundred thousand 020913 GBM stem cells were implanted intracranially in each of the twenty athymic nude mice. The mice were divided into four groups of five each. The treatment groups consisted of mice treated with gefitinib alone, sunitinib alone and combination of gefitinib and sunitinib. The control group consisted of five mice gavaged with phosphate buffered saline (PBS). In order to mimic the human trial, the doses of the drugs administered in animals were equivalent to the FDA approved human doses . The mice in the treatment group were treated with gefitinib alone (75 mg/kg), sunitinib (15 mg/kg) alone and combination of gefitinib (75 mg/kg) and sunitinib (15 mg/kg) three days a week. The median survival of the mice from the control group was 58 days, whereas the mice receiving the RTK inhibitors gefitinib and the RTK combination demonstrated a significantly improved survival (p = 0.0001) with median survival of 94 and 90 days respectively . 060919 cells were generated at Johns Hopkins and were profiled by whole genome sequencing as a part of glioblastoma genome project (sample # BR23X) GBM oncosphere line 020913 was from Sara Piccirillo and Angelo Vescovi, Università degli Studi Bicocca-Millan, Italy. GBM oncosphere lines 060919 and 020913 were cultured in stem/progenitor cell media  containing EGF (20 ng/ml) and FGF (10 ng/ml)  and maintained at 37°C in a humidified incubator with an atmosphere of 5% COp-toluenesulfonate (Nexavar™) were purchased from LC laboratories . MET inhibitor (SU11274), FGFR inhibitors  and PDGFR tyrosine kinase inhibitor III were purchased from Calbiochem . Inhibitors were dissolved in dimethylsulfoxide (DMSO) at a stock concentration of 10 mM or 100 mM and stored at −20°C until used.Gefitinib (Iressa™) and erlotinib (Tarceva™) were kindly provided by Dr. Nisana Namwat . Imatinib mesylate (Gleevec™) was synthesized by American Custom Chemicals Corporation  and supplied by the Ludwig Institute for Cancer Research. Sunitinib malate (Sutent™) and sorafenib Profiling of receptor tyrosine kinases (RTKs), kinases and their protein substrates phosphorylation were analyzed by using Human Phospho-RTK Array Kit  and Human Phospho-Kinase Array Kit  according the manufacturer's instructions using protein extracts from GBM stem-like cell lines (020913 and 060919). Cell lysates were diluted and incubated overnight with the array membrane. The array was washed to remove unbound protein, incubated with an antibody cocktail, and then developed using streptavidin-horseradish peroxidase and chemiluminescent detection reagents.5) were seeded in 6 well plates . After overnight incubation, cells were treated with single drugs and combinations, and then harvested at 24 hours time point. Protein lysates were prepared using RIPA buffer and immunoblot analysis was performed as previously described Cells (2×102) in black clear-bottom 96 well plates (Becton Dickinson) and incubated overnight. The following day, drugs were added as single agents or in combination at designated concentrations and then 20 μl of 10X alamarBlue® was added. The volume in each well was made up to 200 μl with the growth medium. After 72 hours incubation, fluorescence was measured on a Perkin Elmer Wallac 1420 Multilabel counter  with a 540 nm excitation filter and a 590 nm emission filter. Experiments were done twice with 6 replicates for each experiment. For the IC50 calculation, GBM oncosphere cells were treated with RTK inhibitors at nine different concentrations ranging from 100 μM to 1 nM  and DMSO was used as a vehicle control. The DMSO volume was kept uniform at 1% of the total volume. After reading the alamarBlue fluorescence at 72 hours, Graphpad prism 5 was used to calculate the IC50 values. Apoptosis assays were performed using the Caspase-Glo® 3/7 Assay  according to manufacturer's instructions and as described earlier GBM stem-like cell proliferation was assessed using an alamarBlue® assay . GBM oncospheres of the appropriate size range were plated  every 3 to 4 weeks. For intracranial implantation, the 9L gliosarcoma tumor was surgically excised from the carrier animal, cut into 1-mm3 pieces, and placed in sterile 0.9% saline on ice. The excised tumor was then intracranially implanted in 32 F344 female rats weighing 150–200 gms as described previously The 9L gliosarcoma was obtained from Marvin Barker, MD, . For tumor piece implantation, 9L tumor pieces measuring 2 mmAll the animals were treated with drug doses equivalent to FDA approved human doses. A weekly human dose was calculated for gefitinib and sunitinib and was converted to a mouse or a rat dose using the formula prescribed by Reagan-Shaw et al Statistical and graphical analyses were performed using GraphPad Prism 5 . The Student T-test was used to compare the growth inhibition in various groups. Animal survival was analyzed using Kaplan-Meier survival and log rank test.Figure S1Illumina analyses demonstrating amplification of EGFR in 020913 cells.(TIF)Click here for additional data file.Figure S2Fold change in proliferation of 020913 (A) and 060919 (B) cells. 020913 and 060919 cells were treated with FDA-approved RTK inhibitors at 10% of their IC50 concentration. Combination of gefitinib (2 µM) and sunitinib (4 µM) demonstrate increased inhibition in 020913 and 060919 the cell growth compared to other combinations.(TIF)Click here for additional data file.Figure S3RTK combination treatment. 020913 cells were treated with FDA approved and unapproved RTK inhibitors at 25% of their IC50 concentrations.(TIF)Click here for additional data file.Figure S4Vandetanib (Zactima™) combination treatment. 020913 cells were treated with vandetanib in combination with other FDA approved drugs such as imatinib (ima), sunitinib (sun) and sorafenib (sor). Vandetanib is an EGFR and VEGFR inhibitor.(TIF)Click here for additional data file.Figure S5Inhibition of phosphorylation in 020913 cells when treated with a combination of Gefitinib and Sunitinib.(TIF)Click here for additional data file.Table S150 values of RTK inhibitors in GBM oncosphere and adherent cell lines.IC(DOCX)Click here for additional data file.Data S1Calculation of FDA equivalent dose of RTK inhibitors for the animal studies.(XLS)Click here for additional data file.


Recent breakthroughs in preclinical research have highlighted several potential novel therapies for SMA, increasing the need for robust and sensitive clinical trial platforms for evaluating their effectiveness in human patient cohorts. Given that most clinical trials for SMA are likely to involve young children, there is a need for validated molecular biomarkers to assist with monitoring disease progression and establishing the effectiveness of therapies being tested. Proteomics technologies have recently been highlighted as a potentially powerful tool for such biomarker discovery.Spinal muscular atrophy (SMA) is a neuromuscular disease resulting from mutations in the We utilized label-free proteomics to identify individual proteins in pathologically-affected skeletal muscle from SMA mice that report directly on disease status. Quantitative fluorescent western blotting was then used to assess whether protein biomarkers were robustly changed in muscle, skin and blood from another mouse model of SMA, as well as in a small cohort of human SMA patient muscle biopsies.By comparing the protein composition of skeletal muscle in SMA mice at a pre-symptomatic time-point with the muscle proteome at a late-symptomatic time-point we identified increased expression of both Calreticulin and GRP75/Mortalin as robust indicators of disease progression in SMA mice. We report that these protein biomarkers were consistently modified in different mouse models of SMA, as well as across multiple skeletal muscles, and were also measurable in skin biopsies. Furthermore, Calreticulin and GRP75/Mortalin were measurable in muscle biopsy samples from human SMA patients.We conclude that label-free proteomics technology provides a powerful platform for biomarker identification in SMA, revealing Calreticulin and GRP75/Mortalin as peripherally accessible protein biomarkers capable of reporting on disease progression in samples of muscle and skin. Similarly, disease-modifying genes are known to exist that can influence the severity of a patient’s condition [The autosomal recessive disease spinal muscular atrophy (SMA) is caused by deficient expression of full-length survival motor neuron (SMN) protein resulting from disruption to the N1) gene . AlthougN1) gene -9. DiseaMN2 gene ,9. A higondition .SMN1 expression have yielded impressive amelioration in neuromuscular dysfunction and large increases in the lifespan of mice with SMA [SMN2 gene by promoter activation or reduction of alternative splicing of SMN2 exon 7 have also shown therapeutic benefit in animal models [This detailed understanding of SMA genetics has facilitated exciting breakthroughs in pre-clinical research over the past few years, with several approaches indicating significant potential benefits in animal models of the disease. For example, experiments using gene therapy approaches to restore with SMA -14. Othel models -17. As al models .In order to improve the reliability and effectiveness of SMA clinical trials, robust biomarkers are required. Firstly, biomarkers are needed to allow accurate monitoring of disease activity and to predict disease progression in human patients . SecondlProteomics technologies have recently been highlighted as a potentially powerful tool for biomarker discovery . In thisSmn-/-;SMN2tg/tg) was originally obtained from the Jackson Laboratory  and had a mean survival of 5/6 days in our hands. The 'Taiwanese’ SMA mouse model  [Two SMA mouse models were used (both on a congenic FVB background). The 'severe’ SMA mouse model    was alsoMN2tg/0) , giving MN2tg/0) . All aniQuadriceps femoris biopsy samples were obtained, through EuroBioBank [SMN1 gene confirming a genetic diagnosis of SMA. Three age-matched control samples were also obtained, genetically confirmed to have no mutations in the SMN1 gene.oBioBank , from twSmn-/-;SMN2+/+) and wild-type  littermates at postnatal day 1 (P1) and P5 were sacrificed by chilling on ice and decapitation. Levator auris longus  muscles were dissected in oxygenated mammalian physiological saline, as previously described ['Severe’ SMA mice , data were exported from Progenesis as a single mgf file per time-point. These files were then used to identify individual peptide sequences using the Swiss-Prot database via Mascot Daemon (v2.4.0) due to the large file size. As an indication of identification certainty, the false discovery rate for peptide matches above identity threshold was 9.39% for P1 and 3.34% for P5. Protein abundance data per experimental run/sample as an output from the Progenesis software can be found in Additional file P-values presented in Tables Protein was extracted in MEBC buffer . Protein concentration was determined by bicinchoninic acid assay  according to the manufacturer's instructions on solubilised muscle . Then 10 μg aliquots of each muscle type were reduced with 10 mM dithiothreitol and alkylated with 50 mM iodoacetamide prior to digestion with trypsin  overnight at 30°C. Technical replicates (3 × 2.5 μg) of each digested muscle type were injected onto a nano-scale liquid chromatographic tandem mass spectrometry (nLC-MS/MS) system (Ultimate 3000 (Dionex (Thermo Fisher), Hemel Hempstead, UK) coupled to a LTQ Orbitrap XL . The peptides from each digest were separated over a 65 minute linear gradient from 5 to 35% acetonitrile in 0.1% formic acid. The LTQ Orbitrap XL was configured with a TOP 5 methodology comprising a 60 K resolution FT-MS full scan followed by IT-MS/MS scans for the 5 most intense peptide ions. The raw data were then imported into Progenesis LCMS for label free differential analysis and subsequent identification and quantification of relative ion abundance ratios, both up-regulated and down-regulated. Following alignment of MS data, principal component analysis and preliminary filtering , GRP75  or TCP1 beta . Odyssey secondary antibodies were added according to the manufacturer's instructions . Blots were imaged using an Odyssey Infrared Imaging System  at 169 μm resolution. Where possible, each sample was independently run and measured twice to minimise user variability.Protein was extracted from 'Taiwanese’ SMA mouse muscle, skin and blood samples and from human muscle biopsies. Protein levels were quantified by BCA. Quantitative western blots were performed as described previously ,32. BrieP < 0.05 was considered to be significant. Individual statistical tests used are detailed in the results section or figure legends.All data were collected into Microsoft Excel spreadsheets and then analysed using GraphPad Prism software. For all statistical analyses Smn-/-;SMN2+/+;) [In order to identify potential new protein biomarkers capable of reporting directly on the progress of disease in SMA, we utilized unbiased, label-free proteomics technologies to compare the proteome of a pathologically affected tissue in SMA   at earlyMN2+/+;) . The mouMN2+/+;) ,33 and aMN2+/+;) . In ordeSmn+/+;SMN2+/+) at P1 (pre/early-symptomatic) and P5  and proteins were extracted for mass-spectrometry analysis. The raw data from P1 animals was previously analysed using the IPI mouse database and was published [The rostral band of LAL muscle was dissected from 'severe’ SMA mice and littermate controls  or late retention time (more than 72 minutes) were excluded. The runs were then grouped according to the genotype of the mouse  and an ANOVA statistical test was performed to determine whether the means of the two groups were equal. At this stage further stringent filtering was performed and all 1Once the list of candidate peptide ions to identify was created, their MS/MS data were exported to the Swiss-Prot protein database to allow comparison to known peptides, and subsequently proteins. The output of this Swiss-Prot analysis was then re-imported to Progenesis software in order to allow further filtering by excluding peptides that were not associated with more than one protein (conflict resolving). Also, peptides that were improperly cleaved by trypsin were excluded . This led to the identification of 540 proteins in the P5 dataset , but were significantly changed at P5 (late-symptomatic). We therefore took the list of all proteins with modified expression in SMA mice at P5 compared to controls, and searched for expression data for the same proteins in the P1 comparison dataset. Any proteins found to have altered expression at both P5 and P1 were considered to be unsuitable as a biomarker and were therefore removed from the candidate list  of 'Taiwanese’ SMA mice and littermate controls [In order to validate the list of candidate biomarkers generated by our proteomics analysis of skeletal muscle from 'severe’ SMA mice, we wanted to determine whether similar changes in protein levels could be detected in a different skeletal muscle from a genetically unique mouse model of SMA using quantitative fluorescent western blotting. We chose three proteins to validate, based on the magnitude of their expression change and the availability of suitable antibodies for western blotting: Stress-protein 70  and Calreticulin were 1.8- and 1.5-fold up-regulated, respectively, in our proteomic dataset whereas T-complex protein 1 subunit beta (TCP1) was down-regulated by 1.5-fold. We measured levels of these three proteins in the controls .gastrocnemius muscle of 'Taiwanese’ SMA mice and littermate controls at a mid/late-symptomatic time-point . All three patients had a genetic diagnosis of SMA confirmed by a homozygous deletion of the SMN1 gene. Three roughly age-matched control samples were also obtained, genetically confirmed to have no mutations in the SMN1 gene.Next, we wanted to establish whether the increased levels of GRP75/Mortalin and Calreticulin, observed to correlate with disease progression in SMA mouse models, were also measurable in skeletal muscle from human SMA patients. Therefore, we examined levels of GRP75/Mortalin and Calreticulin using quantitative fluorescent western blotting on human muscle biopsy samples obtained through EuroBioBank (see Methods). We obtained biopsies from the Both GRP75/Mortalin and Calreticulin could be readily identified and levels measured using quantitative fluorescent western blotting. Both GRP75/Mortalin and Calreticulin showed a trend towards increased levels in the small cohort of SMA patients compared to controls Figure . GRP75/MOur analyses of GRP75/Mortalin and Calreticulin levels in skeletal muscle from SMA mouse models (supported by preliminary investigations of human patient tissue) suggested that these two proteins may represent robust protein biomarkers for SMA. However, obtaining muscle biopsies from human patients is an invasive procedure that is not ideal for repeated analyses of protein levels during a clinical trial, especially in small children. As a result, the availability of biomarker proteins in more peripherally accessible tissue (such as skin and/or blood) would make it much easier to obtain quick, repeated tissue samples for monitoring purposes. Therefore, we next asked whether GRP75/Mortalin and Calreticulin protein could be reliably identified and measured in skin and blood. Analysis of expression datasets  confirmeFinally, we wanted to establish whether GRP75/Mortalin and Calreticulin levels in skin matched the temporal profile we originally identified in our muscle proteomics experiments. Therefore, we collected skin samples from 'Taiwanese’ SMA mice and littermate controls at four different time-points: P1 and P5 (pre-symptomatic), P7 (early-symptomatic), and P9 (mid/late-symptomatic). Temporal changes in the levels of GRP75/Mortalin and Calreticulin showed similar trends in SMA mouse skin, with no differences observed at pre/early-symptomatic time-points, but robust increases evident after onset of symptoms at P6 Figure C,E. ThusIn this study we used label-free proteomic technology to identify two proteins with the potential to act as molecular biomarkers for SMA. The combination of proteomics technology with an established mouse model of SMA  revealed that increased levels of GRP75/Mortalin and Calreticulin in skeletal muscle correlated with disease progression. Importantly, these protein biomarkers were also accessible in skin samples from SMA mice, suggesting that they can also be monitored in a peripherally accessible tissue during clinical trials. A preliminary study on a small sample of patient muscle biopsies suggested that GRP75/Mortalin and Calreticulin were detectable and measurable in human tissue, including biopsies from SMA patients.The use of label-free proteomics in this study provides further evidence that proteomics technologies represent a powerful tool for biomarker discovery . Indeed,Calreticulin is a multifunctional protein that has previously been identified as a potential biomarker for other diseases. For example, serum levels of Calreticulin have been shown to increase in patients with rheumatoid arthritis  and incrGRP75/Mortalin is a member of the Hsp70 family of chaperones, with roles including the regulation of energy generation, stress responses, muscle activity, mitochondrial activity, and cellular viability -46. As wOur preliminary investigation of GRP75/Mortalin and Calreticulin levels in human skeletal muscle suggests that these proteins may represent viable biomarkers in human SMA patients. However, it represents only an initial demonstration of the ability to detect and measure these proteins in human tissue, and was hampered by a lack of detailed information from the biobank regarding the actual stage of disease progression for each patient at the time of muscle biopsy, as well as the small sample size. As a result, further, large-scale studies on patient cohorts will now be required to validate GRP75/Mortalin and Calreticulin as robust protein biomarkers for SMA in humans. The demonstration in mouse models that increased levels of these proteins correlated with increasing disease severity suggests that such a study is now warranted. Moreover, the finding that these proteins can be tracked in skin samples suggests that the use of skin biopsies might be of more practical use for these studies, reducing the need for repeated invasive muscle biopsies.SMN status of SMA patients, these biomarkers should provide an additional means through which the disease can be monitored and tracked. Further work is now warranted to validate these protein biomarkers in cohorts of SMA patients.We conclude that label-free proteomics technology provides a powerful platform for biomarker identification in SMA. Combining label-free proteomics with established mouse models of SMA led to the identification of Calreticulin and GRP75/Mortalin as protein biomarkers capable of reporting on disease progression in tissue samples of muscle and skin. When used alongside the genetic Levator auris longus; LC: Liquid chromatography; MS/MS: Tandem mass spectrometry; P: Postnatal day; SMA: Spinal muscular atrophy; SMN: Survival motor neuron.LAL: The authors declare that they have no competing interests.CAM, TMW and THG designed the study; CAM DJL, GH, TMW and THG performed experiments and analysed data; CAM, TMW and THG wrote the manuscript; all authors read and approved the final manuscript.Protein abundance output from Progenesis analysis software for all identified proteins from P5 SMA versus control animals.Click here for filePeptide abundance (feature) output from Progenesis analysis software for proteins from P5 SMA versus control animals.Click here for file