text:
- Research article
- Open Access
Characterization of the doublesex gene within the Culex pipiens complex suggests regulatory plasticity at the base of the mosquito sex determination cascade
- Dana C. Price1Email author,
- Andrea Egizi1, 2 and
- Dina M. Fonseca1
© Price et al. 2015
- Received: 24 November 2014
- Accepted: 22 May 2015
- Published: 11 June 2015
The doublesex gene controls somatic sexual differentiation of many metazoan species, including the malaria mosquito Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti (Diptera: Culicidae). As in other studied dipteran dsx homologs, the gene maintains functionality via evolutionarily conserved protein domains and sex-specific alternative splicing. The upstream factors that regulate splicing of dsx and the manner in which they do so however remain variable even among closely related organisms. As the induction of sex ratio biases is a central mode of action in many emerging molecular insecticides, it is imperative to elucidate as much of the sex determination pathway as possible in the mosquito disease vectors.
Here we report the full-length gene sequence of the doublesex gene in Culex quinquefasciatus (Cxqdsx) and its male and female-specific isoforms. Cxqdsx maintains characteristics possibly derived in the Culicinae and present in the Aedes aegypti dsx gene (Aeadsx) such as gain of exon 3b and the presence of Rbp1 cis-regulatory binding sites, and also retains presumably ancestral attributes present in Anopheles gambiae such as maintenance of a singular female-specific exon 5. Unlike in Aedes aegypti, we find no evidence for intron gain in the female transcript(s), yet recover a second female isoform generated via selection of an alternate splice donor. Utilizing next-gen sequence (NGS) data, we complete the Aeadsx gene model and identify a putative core promoter region in both Aeadsx and Cxqdsx. Also utilizing NGS data, we construct a full-length gene sequence for the dsx homolog of the northern house mosquito Culex pipiens form pipiens (Cxpipdsx). Analysis of peptide evolutionary rates between Cxqdsx and Cxpipdsx (both members of the Culex pipiens complex) shows the male-specific portion of the transcript to have evolved rapidly with respect to female-specific and common regions.
As in other studied insects, doublesex maintains sex-specific splicing and conserved doublesex/mab-3 domains in the mosquitoes Culex quinquefasciatus and Cx. pipiens. The cis-regulated splicing of Cxqdsx does not appear to follow either currently described mosquito model (for An. gambiae and Ae. aegypti); each of the three mosquito genera exhibit evidence of unique cis-regulatory mechanisms. The male-specific dsx terminus exhibits rapid peptide evolutionary rates, even among closely related sibling species.
- doublesex
- mab-3
- Culex quinquefasciatus
- Culex pipiens
- Sterile insect technique
- Vector biology
- Sex determination
The manifestation of distinct sexes is fundamentally conserved among most metazoans. However, the development of sex-specific somatic and gonadal tissues and neuronal processes (e.g. behaviors) is governed by a variety of factors both environmental and genetic, and often varying widely between and within taxa [1–3]. Most animals direct sex-specific cell fate by function of the Doublesex/Mab-3 Related Transcription factor (DMRT) family of zinc-finger proteins [4, 5] and the genes they regulate. Within the insects, this process involves a genetic cascade first elucidated in the model fly Drosophila melanogaster [6] whereby a primary signal triggers sex-specific splicing of one or more regulatory factors which subsequently bind pre-mRNA of the conserved DMRT "major switch" gene, doublesex, and direct its sex-specific splicing, thus initiating development of male or female forms [7].
Although there are many diverse primary signals that initiate the cascade (e.g. X:A ratio, M-factors, W/Y chromosomes; see [1]), dsx appears to be conserved as the major switch at the base of the cascade [8, 9]. In many insects the male and female-specific splicing of dsx is directed by the upstream regulator transformer, a serine/arginine rich (SR) protein which itself is transcribed in a sex-specific manner, as well as the constitutively expressed transformer-2 [10, 11]. The resultant TRA/TRA2 peptide complex binds the dsx mRNA at the dsx repeat element (dsxRE), facilitated by the purine-rich enhancer (PRE) element [12, 13], and directs sex-specific splicing of dsx mRNA for translation into male (DSXM) or female (DSXF) peptides. In Drosophila, an additional SR splicing enhancer component, RBP1, binds to target sites in the splice acceptor preceding the female-specific exon and is essential for efficient splicing of female dsx pre-mRNA [14]. The downstream targets of insect dsx are not well elucidated, however 58 optimal binding sites and associated nearest genes have been identified for D. melanogaster Dmdsx [15]. The red flour beetle Tribolium castaneum Tcdsx has been implicated in oocyte development including Vitellogenins and their associated receptors [16], while Lepidopteran dsx has been shown to influence expression of pheromone-binding proteins and hexamerin storage proteins [17].
Orthologs of the dsx gene have currently been identified in seven orders of insects ranging from the primitive Pediculus humanus (human body louse) to several genera of Hymenoptera, however a functional transformer homolog has not always been recovered in these genomes (see [8] for summary) leading to speculation that some lineages have recruited alternate or additional upstream regulators for dsx [18]. For example, TRA/TRA-2 mediated splicing of dsx has been shown in the Brachyceran flies Ceratitis capitata [19], Musca domestica [20] and Lucilia cuprina [21] yet transformer appears lost in the Nematoceran flies including mosquitoes [8].
Despite varying primary signals and upstream regulatory mechanisms, male and female-specific DSX peptides of various Diptera including Anastrepha [22], Ceratitis [23] and Musca [24] effected partial masculinization and feminization of genetically female and male D. melanogaster, respectively, when expressed ectopically. This evolutionary conservation is due in part to the retention of two functional protein domains essential for peptide oligomerization: an atypical zinc-finger DNA-binding domain found in multiple members of the DMRT superfamily (DBD/OD1) and an oligomerization domain (OD2) unique to dsx [25]. The DBD/OD1 domain functions to form a dimeric DNA-binding unit that maintains 92 % sequence similarity between Dipteran (D. melanogaster) and Lepidopteran (Bombyx mori) taxa while completely conserving the critical cysteine and histidine residues [26]. The OD2 domain is likely responsible for sex-specific splicing activation or repression of downstream factors [25], and is modified by sex-specific splicing to maintain both common and male/female-specific portions; the common portion exhibits a greater degree of conservation within and among insect taxa than the C-terminal sex-specific portion [18, 26].
The current Aeadsx gene model [18] (Fig. 1) spans 450 kb of genomic DNA of supercontig 1.370 and is composed of eight known exons, although nine are likely. Unlike other sequenced Dipteran dsx genes, Aeadsx was found to produce two female-specific isoforms by exon skipping, encoding peptides with alternative C-termini via inclusion of both exons 5a and 5b, or 5b alone. Additionally, analysis of cis-acting elements in Aeadsx revealed a cluster of TRA-2-ISS and RBP1 elements upstream of exon 5a, and Dipteran dsxRE binding sites and PRE elements present only in exon 5b (Fig. 1). Several instances of a motif strongly resembling a potential dsxRE element previously only recovered in the Hymenoptera (NvdsxRE, [11]) were found within exon and intron 5a. Unlike An. gambiae (and similar to Drosophila) Aeadsx possesses a weak splice acceptor upstream of exon 5b that is activated to splice both female isoforms. Salvemini et al. [18] hypothesize that regulatory mechanisms governing the sex-specific splicing of the gene in Ae. aegypti are different than in other Diptera including An. gambiae, and that the two female-specific exons were each under the control of a different splicing regulator: A female-specific TRA-like protein acts in females as a splicing activator of exon 5b via dsxRE and PRE elements, while a splice repressor acts on 5a (included by default splicing) in some transcripts. In the males, a male-specific factor may act to repress inclusion of exon 5a via TRA-2-ISS and NvdsxRE elements, while exon 5b is excluded due to lack of female-specific TRA.
Cho et al. [29] proposed that default female-specific dsx splicing by selective repression of the male isoform (i.e. by the feminizer gene in A. mellifera [30] and the recently discovered piRNA precursor Fem in B. mori [31]) is ancestral to holometabolous insects based on its conservation in taxa as phylogenetically distant as A. mellifera and B. mori, and that Diptera possess a derived splicing system where the male form is default and the female form must be 'splice-activated' by a TRA/TRA2-like factor. While this appears to be the case in Anastrepha, Drosophila, and An. gambiae doublesex, the data from Salvemini et al. [18] strongly suggest that the female spliceforms are default in Ae. aegypti; the "strong" exon 5a does not require TRA/TRA2 enhancement, and must be repressed by a male factor. Culicine mosquitoes (inclusive of the genera Aedes and Culex) determine sex at an autosomal locus [32], while Anopheline mosquitoes possess heteromorphic (XY) sex chromosomes [33]. The latter authors propose that this locus (the M-locus) may either act on intermediary factors or on the dsx gene itself (transformer appears to be either lost or extremely diverged in the mosquitoes [8], however transformer2 is present) to suppress female-specific dsx splicing and generate the male form. Further, Salvemini et al. [18] posit that retention of the Hymenopteran-like NvdsxRE elements coupled with Apis-like splicing regulation (and a likely female-specific default splicing) could represent a stably maintained ancestral state in Ae. aegypti exclusive of the rest of known Dipteran doublesex. Recently, analysis of the red flour beetle Tribolium castaneum [34] revealed three female-specific and one male-specific dsx isoform, with male default splicing occurring via suppression of maternally transferred zygotic TRA protein (required to activate female-specific splicing) by a dominant male factor. This variation in the top-level regulation of dsx among Hymenoptera, Diptera, Lepidoptera, and Coleoptera via upstream factors is in agreement with the theory of Wilkins [35] stating that the cascade has evolved in reverse order, with the final double-switch gene (doublesex) remaining relatively conserved as additional elements are added and/or neofunctionalization occurs at the upper regulatory levels. As sex determination is critical to insect reproduction, deleterious mutations in dsx could therefore have strong effects on fitness and be selected against. Previous studies have shown the female-specific exon to be evolutionarily conserved [36–38], yet disagree on evolutionary rate comparisons of the common and male-specific portions of the transcript over longer evolutionary time frames. Hughes [38] found a much greater rate of non-synonymous substitutions within the male-specific region as compared to the common region, while Sobrinho Jr. and de Brito [39] found nearly equivalent levels of positive selection between the two.
As the production of genetic sexing mosquito strains and molecular methods that create male bias and/or elimination of the female sex are ideal strategies for sterile insect technique [40], it follows that a conserved sex regulator like doublesex (and transformer) would be optimal molecular targets for such control programs [41]. Elucidating the variable mechanisms by which dsx determines sexual fate in sequenced mosquito lineages is mandatory if progress is to be made towards a control strategy for the world's deadliest animals. Here we provide full-length gene sequence, sex-specific splicing analyses, and regulatory analysis of the doublesex gene from the southern house mosquito Culex quinquefasciatus (herein Cxqdsx) via RT-PCR and Illumina transcriptome data. Additionally, to discern the strength and location of early evolutionary drivers on doublesex within the Culex pipiens complex, we conduct an evolutionary analysis using Cxqdsx and a newly constructed dsx transcript from Culex pipiens form pipiens (Cxpipdsx). These results provide a comparative platform with which to study sex determination in those mosquitoes with currently sequenced genomes (An. gambiae [42], Ae. aegypti [43] and Cx. quinquefasciatus [44]).
Culex quinquefasciatus mosquitoes were obtained from a colony initiated in 2008 with egg rafts collected from Oahu, Hawaii, USA. Male and female total RNA was extracted separately from twenty adult mosquitoes of each sex using the Qiagen RNeasy Plus Universal Kit (Qiagen, Valencia CA) per manufacturer's protocol. Prior to extraction, samples were placed in a 2 ml eppendorf tube containing a sterile steel bead + 800 μl Qiazol solution and homogenized for 1 min @ 20Hz on a Qiagen TissueLyser. Contaminant DNA was removed with the TURBO DNA-free DNA Removal Kit (Invitrogen, Carlsbad CA) and first-strand cDNA was generated using the Superscript First-Strand Synthesis System (Invitrogen) per manufacturer's protocol and diluted to 50 μl in H2O. Four microliters of the cDNA was used in each 25 μl PCR reaction containing 12 μl H2O, 2.5 μl Qiagen Q-solution, 2.5 μl 10× PCR buffer, 0.5 μl dNTPs, 2.5 units AmpliTaq DNA Polymerase (Invitrogen) and 0.5 μl (200 μM final concentration) of each primer. Thermal cycling conditions were as follows: 1 min @ 95 °C, followed by 30 cycles x (30 s @ 94 °C, 30 s @ 50-54 °C primer-specific annealing, 60 s @ 68 °C [120 s for products > 1 kb]), 5 min @ 68 °C final extension.
To recover the complete 5' end of the transcript, we performed 5' RACE PCR using the FirstChoice RLM-RACE Kit (Invitrogen) per manufacturer's protocol using internal gene-specific primers quinqDSX5RACE-GSP1 and quinqDSX5RACE-GSP2 placed adjacent to the OD1 domain. All RT-PCR and RACE-PCR amplicon products were visualized on a 1.5 % agarose gel in TAE buffer and gel-purified using the QIAquick Gel Extraction Kit (Qiagen) prior to cloning via the TOPO TA Cloning Kit (Invitrogen) and PCR-enrichment using the M13 forward/reverse primer pair per manufacturer's protocol. PCR products were cleaned with ExoSap (Invitrogen) per manufacturer's protocol, and cycle sequencing was performed by GENEWIZ (South Plainfield, NJ) using the M13 primer pair.
The 3' end of Cxqdsx was predicted, and the entire gene sequence qualified by mapping the paired-end RNAseq data from NCBI SRA accession SRR991016 generated by Leal et al. [45] to Cx. quinquefasciatus supercontig 3.59 using the CLC Genomics Workbench (CLC Bio, Aarhus, Denmark) large-gap read mapper (nucleotide similarity score of 95 % over a 95 % read length fraction) and manually examining the output. This process was repeated using the Cx. pipiens f. pipiens paired-end RNAseq library generated by [46] (see Additional file 2 for details) and the Cx. quinquefasciatus reference generated above to create the full-length gene structure for Cx. pipiens f. pipiens doublesex (Cxpipdsx). To extend the gene model for Aeadsx, we repeated this protocol yet again with the Ae. aegypti NCBI short-read paired-end libraries SRR924024 and SRR789758 and AaegL1.4 supercontig 1.370.
To assess the distribution of the consensus dsxRE (TRA/TRA2) and RBP1 type-b motifs (derived from those of D. melanogaster, An. gambiae and Ae. aegypti), we screened all transcript coding (CDS) sequences corresponding with the Cx. quinquefasciatus Cpip1.3 dataset from VectorBase for their presence. The degenerate motif was broken down into all possible constituents, and each was queried against the CDS dataset with BLASTn (e-val = 999, word_size = 13 [dsxRE] or 7 [RBP1b]). The output was parsed via custom Perl scripts, and transcripts containing six copies of the motif in a 224 bp (for the dsxRE; 546 bp for RBP1b) window were retained. The AhoPro software utility [47] was used to calculate the probability of observing the motif against a reference dataset of nucleotides randomly generated under a Bernoulli/0-order Markov model.
The synonymous substitutions per synonymous site and nonsynonymous substitutions per nonsynonymous site (Ks and Ka, respectively) and the Ka/Ks ratio were calculated in a pairwise comparison between Cxqdsx and Cxpipdsx using the KaKs Calculator v2.0 [48] under model averaging (MA). We re-calculated these values for each sliding 30 bp window while moving 3 bp (1 amino acid) downstream at a time. To examine base composition of splice acceptor sites, we retrieved 52,278 internal (i.e. exclusive of exon 1) exons with 16 nt of upstream sequence from the CpipJ 1.3 assembly (Vectorbase, [49]) and calculated the mean number of pyrimidines in the 12 nt preceding the 4 nt splice acceptor.
Results and discussion
Structure and splicing of Cxqdsx
To qualify our Cxqdsx gene model, we mapped the short-read Illumina RNAseq data in NCBI SRA accession SRR991016 generated by Leal et al. [45] to supercontig 3.59 and manually annotated Cxqdsx. The transcript was well represented in these data, and the structure congrued with our RT-PCR and 5'RACE results in the placement and splicing of all previously described exons including the lack of additional spliceforms in female-specific exon 5 as well as the sequenced 5' common end of exon 6. Additionally, these data allowed us to define the C-terminus of Cxqdsx, including the full 1,016 bp male-specific/common exon 6 and its splicing over 13,814 bp of intron to a terminal 2,201 bp 7th exon/UTR (Fig. 1, Additional file 3: Figure S1). The final Cxqdsx protein product (Fig. 3) initiates translation in both females and males from the start codon in the common exon 2, and terminates in female mosquitoes at the opal-ochre double stop codons (conserved in Diptera, see [51]) within exon 5 and in male mosquitoes at a stop codon within exon 6. Exons 6 and 7 are thus transcribed entirely as UTR in the female isoform, as has been shown in other Dipterans including Megaselia scalaris [51], Anopheles gambiae [27] and Aedes aegypti [18].
Completing the Aeadsx gene
To compare the size, structure, intron characteristics and putative promoter regions of our full-length gene model with that of the other sequenced Culicine mosquito, Ae. aegypti, we used publicly available Illumina short-read RNAseq data to discern in-silico the 5'UTR, transcription start site, exon 1 and full 3'UTR of Aeadsx [18]. To predict the 5' end of Aeadsx, we mapped Illumina short-read RNAseq libraries from NCBI SRA accession SRR789758 to Aedes aegypti strain Liverpool supercontig 1.370 as performed previously and located exon 2 defined by Salvemini et al. [18]. By visual inspection of the mapping, we were able to extend the 2nd exon 472 bp upstream of the start codon, define a splice junction spanning 14,481 bp of intronic sequence, and locate a 1,388 bp 1st exon/5'UTR (Fig. 1, Additional file 5: Figure S3). As RNAseq mapping provides only approximate definition of transcript ends, we searched for a promoter motif within an area +/− 250 bp from the point at which 5' short-read coverage for exon 1 ceased. We located an initiator element (Inr) of the form YYANWYY at position 109460 of the reverse-complemented supercontig 1.370 and a downstream promoter element (DPE) of the form RGWYV at canonical position +28 from the Inr adenine (Fig. 5), thus providing strong evidence for the Aeadsx transcription start site. As in Cxqdsx, no TATA box was found.
Splice donors and acceptors of the Cxqdsx gene. Coding (exon) sequences are in uppercase text, while the splice donor/acceptor and succeeding/preceeding 12 nucleotides, respectively, are in lowercase. "Exon 4ex" denotes the alternate downstream splice donor of exon 4. Asterisk indicates the splice acceptor site deviates significantly from the genomic mean of 8.58 +/− 1.39 SE (see Methods)
exon end
splice donor
splice acceptor
next exon begin
No. purines
exon 1
intron 1
exon 2
intron 2
exon 3a
intron 3a
exon 3b
intron 3b
exon 4
intron 4
exon 4ex
intron 4ex
exon 5
intron 5
exon 6
intron 6
Repetitive elements
The genera Aedes and Culex are estimated to have diverged ca. 52 Mya [44]. The genome size for Cx. quinquefasciatus currently stands at 540Mbp [44], while that of Ae. aegypti is estimated to be over twice that size at 1.3Gbp, largely due to the accumulation of transposable elements (TEs) [43]. As TEs are not distributed randomly within chromosomes [52, 53], we assessed the frequency of repetitive elements within the doublesex gene in order to determine whether different classes have invaded the respective dsx genes of Cx. quinquefasciatus and Ae. aegypti. We used CENSOR (http://www.girinst.org/censor/index.php) to scan Cxqdsx introns 2–7 and compared the results to those for Aeadsx intron 2–8 [18] (Additional file 8: Table S2, Additional file 9: Table S3). The two genes contain nearly identical numbers of DNA transposons and similar numbers of LTR retrotransposons, however Aeadsx was found to contain nearly twice as many Non-LTR retrotransposons (or LINEs). These elements persist with great success in eukaryote genomes [54] and comprise 4 % and 14 % of the transposable elements in the Cx. quinquefasciatus and Ae. aegypti genomes, respectively [43, 44], thus their abundance in doublesex likely reflects the genome-wide pattern.
Regulatory mechanisms of Cxqdsx
All splice junctions of Cxqdsx use conserved GT-AG splice donor/acceptor motifs (Table 1). Interestingly, we find that the number of purines in the polypyrimidine tract of the 3' splice acceptor preceding the common/male-specific exon 6 (n = 5) deviates significantly from the calculated mean (8.58, +/− 1.39 SE, see Methods) and constitutes a suboptimal splice acceptor. This is contrary to Aeadsx, which is hypothesized to activate a weak splice acceptor upstream of the female-specific exon 5b [18], and Angdsx which likely relies on activation of the 5' weak splice donor downstream of exon 5 [27].
Twenty-two copies of an RBP1 type B motif were present; fourteen copies were located outside of exon 6, however these were represented by eleven different permutations of the consensus sequence. Each 7 nt permutation had a BLASTn e-value of 1.3 when queried against the full Cxqdsx gene sequence, and (in the absence of a clustered distribution) can be expected to occur at least once by chance. Eight copies, however, were clustered in a 546 bp stretch at the 5' end of the male-specific exon 6. Repeating the protocol used in the TRA/TRA2-like enrichment test above, we find 86 of 19,019 transcripts (0.45 %) contain 8 or more copies of the RBP1b consensus in a 546 bp window. Many of these contigs generated positive results due to tandem repeats however (data summarized in Additional file 12: Table S5). Using AhoPro [47], we determined the probability of observing this motif in 546 bp of randomly-generated sequence data to be 2.89x10−5. A cluster of Rbp1 binding sites and TRA-2-ISS elements upstream of the "strong" female-specific exon 5a of Aeadsx are hypothesized to manage the differential splicing of this exon while other TRA/TRA2-like elements enhance the "weak" exon 5b [18] (see Fig. 1). The localization of this RBP1-binding cluster near the "weak" or suboptimal splice acceptor in Cxqdsx exon 6 indicates a SR-like factor may be involved in its splicing. This presents a curious model, as exon 6 is included in both male and female spliceforms. It is thus likely that if exon 6 requires activation by a SR-like factor, it would occur in the male-specific spliceform and facilitate excision of the female-specific exon 5. This would require use of the exon 6 splice acceptor at the expense of exon 5, and could be facilitated by the Rbp1 elements. The functional TRA/TRA2-like factor present in the female would then suffice to maintain incorporation of exon 6 as UTR. Five copies of the TRA-2-ISS motif were found but were not in significant representation. Three copies of the NvTRA element were found, however unlike in Aeadsx that maintains four copies within a cluster in exon 5, two copies were found in intron 4 and one in exon 5. The BLASTn e-value of each 8 bp hit within the search area was 0.37, thus we cannot exclude this result as having occurred simply by chance.
Sequence evolution of Cxqdsx
Our results show that the Cx. quinquefasciatus doublesex gene exhibits sex-specific splicing, as it does in the mosquitoes Ae. aegypti and An. gambie, as well as in other Diptera. Cxqdsx shares characteristics of both Aeadsx (gain of exon 3b, Rbp1 cis-regulatory binding sites) and Angdsx (singular female-specific exon, shared 3' UTR), as well as a novel spliceform generated from an alternate exon 4 splice donor that appears to occur only in the female. Additionally, we complete the full-length Aeadsx model and identity a putative TATA-less Inr/DPE core promoter region in both Cx. quinquefasciatus and Ae. aegypti mosquito genomes, allowing for future in situ validation and studies of dsx gene transcription.
We find that cis-regulatory splicing regulation of Cxqdsx does not appear to follow either currently described mosquito model, and instead involves activation of a weak splice acceptor of the male-specific/common exon 6, possibly involving a cluster of local Rbp1 binding sites as enhancers. This finding further exemplifies the diversity present in upstream splicing regulation of dsx within mosquitoes, as each of the three genera studied (Anopheles, Aedes and Culex) possess unique regulatory mechanisms despite maintaining TRA/TRA2-like binding sites in the 3' end of their respective female-specific exons (exon 5b in Aeadsx).
An analysis of peptide evolutionary rates between Cxqdsx and the dsx gene of the closely related Cx. pipiens form pipiens (Cxpipdsx, also generated in this study) shows that the male-specific component of the transcript has evolved at accelerated evolutionary rates relative to the female isoform, and contains sites exhibiting signs of positive selection. This result accentuates the rapid evolution of doublesex within the Culex species complex. Future research defining the degree to which doublesex influences the sexual selection cycle may shed light on the role (if any) that this integral gene plays in incipient speciation within insects.
Availability of supporting data
The nucleotide sequences for the male and female-specific Cxqdsx transcripts have been submitted to GenBank under accession numbers KP033512 and KP033513, respectively. Sequences for male and female-specific Cxpipdsx transcripts have been submitted under accession numbers KP033514 and KP033515.
We are grateful to Linda McCuiston for her unsurpassed expertise in rearing and colonizing the mosquitoes used in our study and to Nicole Wagner at the Rutgers University School of Environmental and Biological Sciences Genome Cooperative for performing our Illumina Sequencing. This work was funded by a New Jersey Mosquito Control Association Daniel M. Jobbins scholarship to DCP and by NE-1043 Multistate funds to DMF.
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This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.


text:
Windows Repair Pro All In One
Windows Repair is an all-in-one repair tool to help fix a large majority of known Windows problems including registry errors and file permissions as well as issues with Internet Explorer, Windows Update, Windows Firewall and more. Malware and installed programs can modify your default settings. Using Tweaking.com – Windows Repair you will have the possibility to restore your OS to its original settings.
Reset Registry Permissions
- Reset File Permissions
- Register System Files
- Repair WMI
- Repair Windows Firewall
- Repair Internet Explorer
- Repair MDAC & MS Jet
- Repair Hosts File
- Remove Policies Set By Infections
How to crack ?
- Download & Install
- copy cracked files to install directory
- Enjoy
Download Link Given Below
Windows Repair Pro All in one with crack Mirror

subset: commoncrawl


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Nothing reminds me of grandma's house more than a piping hot chicken pot pie, fresh from the oven. Grandma's chicken pot pies always had a golden brown layer of the most perfect flaky pastry. It was so much fun to break into the crust with a fork (or a spoon), to reveal tender bites of chicken, perfectly cooked veggies, and a sauce so good I could drink it with a straw. Does it get any better than that?
I still crave the comfort of a chicken pot pie — especially as the weather gets colder. Digging into this dish is like a warm hug on a cool evening. If only all my hugs could come in the form of fresh pies! And better yet, this recipe is a total crowd-pleaser, so I know everyone will be licking their plate clean.
Grandma used to spend hours building the most tender pastry crusts. She'd do them in large batches — making plenty of freezer-ready savory and sweet pies for the month. It was a full-day activity for her. I take a clever shortcut (sorry grandma!). The end result is just as delicious, but takes a fraction of the time. Which means healthy, home-cooked dinners on a weeknight. And, did I mention this shortcut also significantly cuts down on the fat?
Phyllo dough is the perfect weeknight trick to getting a tender, flaky crust for your chicken pot pie with minimal effort. Phyllo dough (also look for spelt filo dough) is typically sold frozen. All you need to do is let it defrost in the fridge overnight, or if you forget, leave it out on the counter to thaw for an hour or two before making dinner. Unlike puff pastry or traditional pie crusts, phyllo has a lot less fat in the dough. To build up layers, you'll brush the paper-thin sheets with a small amount of olive oil to help them stick together as they bake. You'll still get all the satisfaction of a crispy, flaky crust — just without gobs of butter. Count me in. As an added bonus, you'll have plenty of phyllo sheets leftover from this recipe, which you can save for another chicken pot pie (or two) in the coming weeks. Once you taste this dish, you'll definitely want to make it again and again!
Chicken Pot Pie
You'll want to dig into this hearty Chicken Pot Pie in the winter months!
- 6 tsp. olive oil divided use
- 1 medium onion chopped
- 2 medium carrots chopped
- 4 oz. sliced mushrooms
- 2 Tbsp. whole wheat flour
- 1½ cups reduced fat (2%) milk
- 1 cup low-sodium organic chicken broth
- ½ tsp. sea salt or Himalayan salt
- ½ tsp. ground black pepper
- 1 tsp. finely chopped fresh thyme
- ¾ cup frozen green peas
- ¾ cup cubed cooked sweet potatoes
- 2¼ cups cubed cooked chicken breast approx. 12 oz.
- 6 sheets phyllo dough (each approx. 12 x 17-inches)thawed, covered in a slightly damp towel
Preheat oven to 375° F.
Heat 2 tsp. oil in large nonstick skillet over medium heat.
Add onion and carrots; cook, stirring frequently, for 4 to 5 minutes, or until onion is translucent.
Add mushrooms; cook, stirring frequently, for 4 to 6 minutes, or until mushrooms are soft and most of the liquid has evaporated.
Add 1 tsp. oil; cook, stirring frequently, for 1 minute.
Add flour; cook, stirring frequently, for 1 to 2 minutes, or until flour and oil form a paste.
Slowly add milk, broth, salt, pepper, and thyme. Bring to a boil, stirring frequently. Reduce heat to medium-low.
Add peas, sweet potatoes, and chicken; cook, stirring frequently, for 2 minutes.
Place chicken mixture in a 2½-quart baking dish. Set aside.
Place phyllo on work surface. Keeping the original stack covered, remove one sheet at a time and quickly brush with remaining 3 tsp. oil. Cut oiled stack of phyllo into quarters. Place phyllo loosely on top of chicken mixture; covering evenly.
Bake for 30 to 35 minutes, or until chicken mixture is bubbling and phyllo dough is golden brown.
Recipe Notes
The Nutrition Facts box below provides estimated nutritional information for this recipe.
Photographs by Anguel Dimov


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50 Hour Service Challenge 2018-2019: Log Your Service Hours
Thank you for contributing to the greater community through your volunteer efforts! Please take a moment to share your experience(s) and log your hours!
50 Community Service Challenge (September 1 - April 1) - those who complete the challenge will be recognized at the LIFE Awards in April, receive a free t-shirt, and entered to win a prize.
Reasons to log your volunteer hours through our office include:
- Getting recognized for your service to the community, whether it be through your class, department, school, residence hall, Greek organization, and/or student organization.
- Discovering new opportunities for becoming involved in service.
- Maintaining accurate and verified records of your volunteer hours. You'll be more competitive for fellowships, scholarships, and graduate school applications.
- The ability to obtain an official record of service through our office upon request.
- Did you volunteer with a student organization? Be sure to also complete the accomplishments form for the SOuRCe!
Feel free to contact us with any questions at firstname.lastname@example.com or at 330.972.2837
This form closed on May 3, 2019 11:59 PM EDT


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I've been noshing on these gluten-free, vegan nutrition bars at various race expos for quite a while now, and now I'm finally writing about them!
Besides being free of the commonly found whey that both disgusts and sickens me and the artificial sugars that taste sub-par and bloat me, these bars are high-protein (~14 g per bar, with ~230 calories) and all-natural.
So far, I have tried the chocolate, almond, strawberry, and peanut butter flavors. I actually like all of them. The flavors are there but not overpowering (particularly in the chocolate one). The texture of these bars is chewy -- you won't find a lot of huge chunks of anything in them. The moisture level is on the low side, but the bar is not crumbly at all.
I really like these bars. I haven't been purchasing too many bars lately, so I'm not sure if I will be getting some unless I need a quick meal and they happen to be around. Additionally, I'm not sure if they are widely available, but if you see them, you should give 'em a try.


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Cointreau 1L
Cointreau is the world's first Triple Sec . Made from sweet and bitter orange peels, it's unique recipe retains only the best natural orange aromas.
The original, the best. Perfect base for cocktails, with limitless versatility. 100% natural ingredients.
A perfect blend of sweet and bitter orange peels, for a unique balance of zesty,fresh and fruity notes.
Try in Cointreau Fizz
purified water ethyl alcohol from agricultural origin sugar (sucrose) vegetable substances (sweet an bitter orange peels)
Pick up the goods you have chosen at Chop Tysa border-shop "Duty Free Shop". You need to give your pre-choice's number or your name to collect your purchase.


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Vivint Classic 4+
- Free
Vivint, Inc. is one of the largest home automation companies in North America. Operating from offices throughout the United States and Canada, the company services close to 500,000 customers. Vivint is dedicated to enhancing security, convenience and energy efficiency. We do this through a combination of award-winning customer service and smart technology. The Vivint Go!Control panel creates a streamlined network that connects all of your home's smart systems: security, HVAC, lighting, small appliances, video, and others. In addition, with consecutive awards from J.D. Power and Associates, as well as recognition from other internationally respected organizations, we are proving our commitment to providing an exceptional customer experience.
What's New
Version 3.2.9
• Minor fixes and enhancements
Ratings and Reviews
**App Feature Request** avoided potential break-in!
This app is great for notifying me when someone approaches my front door. Just today, a stranger approaches my front door, knocks on it, and then checks the door knob and walks off! It would be great to have recorded more than 30 seconds of his activity. I can only see him walking up to the door from his truck nor can I see him getting back into his truck. Luckily I was able to check my phone while I was at work and watched him walk around the side of my house and then leave; he was in and out in under 40 seconds. Please allow us to either record 24/7 or to extend the motion detected recording time for; 45s, 60s, 90s, and allow us to manually turn on video recording when we see suspicious activity taking place. More great features would be an emergency contact option like the ones on the sky panel and local weather like on the sky panel. Also, I found a bug in the app that caused me not to be able to speak to this potential criminal because the microphone button was not visible, it might have something to do with my wife viewing the same camera live at the same time. Thank you so much Vivint!!!
Great app but....
the actual monitoring is a different story. App is simple and user friendly but does lack any of the options or functionality the the wall unit has. In fact i find the app is easier to use than the wall monitoring unit or control panel and think that the panel should be eliminated since it only gives a perpetrator something to either break or make the homeowner deactivate. Something much harder to do since the perp would not want the homeowner to even have their phone when committing the crime and most likely take the phone away so turning the alarm off would be impossible to do. Service from the company on the other hand is not impressive as i have yet to ever get a follow up call once the alarm has been tripped. So if your looking for an unmonitored alarm system with some pretty good modern technology then this system is right for you. Keep in mind it will be extremely expensive since this "unmonitored" system still comes at a monitored price with payment due every month for like ten years! Seriously.
Vivint App
This app works flawlessly. In response to the one user's statement about lag when setting from the car: this is a wifi issue and not the app. I will notice this from time to time myself when arming for the night with the app and the delay is ALWAYS due to wifi needing refreshed. The panel is "online" itself and not on private wifi. Changing to cellular data on your mobile device, especially if you're in the fringe zone of your wifi coverage, and giving your mobile device a second or two to catch its breath will overcome the extensive delay mentioned. Sometimes rebooting my mobile device is the cure. Technology is a wonderful thing if you can truly harness its abilities.
App works as advertised and without issues. Keep in mind, the issue isn't with your system per say, you'll find proper wifi/cell data use will enhance your experience. I'm not just a Vivint user, I also work directly with wifi/data connections in my profession.
- Seller
- Vivint, Inc.
- Size
- Category
- Lifestyle
- Compatibility
Requires iOS 6.0 or later. Compatible with iPhone, iPad, and iPod touch.
- Languages
English, French, Portuguese, Spanish, Turkish
- Age Rating
- Rated 4+
- Copyright
- © 2012 Vivint, Inc.
- Price
- Free
Family Sharing
With Family Sharing set up, up to six family members can use this app.


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Big Event Photography
Contact us
Balls, Proms and parties
Corporate Events, conferences and product launches
Sports and motor sport photography
Equestrian events photography
Big Event Photography Blog
Apply to work as an event photographer with Big Event Photography
Big Event Photography
(and the smaller ones too!)
Big Event Photography provide event photography throughout Scotland providing corporate and private events with photography solutions that meet their needs.
Who are we?
Duncan has almost a decade of experience and works with a team of specialist photographers to allow him to scale his coverage to any event. We are very customer focused and this is an ethos instilled throughout our team, ensuring that all the people that step in front of the camera have a fun experience and get the most out of their event photographs.
What do we do?
As an expert event photographer Duncan, with his team, offers a range of services for your events, instant onsite prints, secure digital galleries, an onsite studio and other specialist services. Any additional photographers brought along to cover your event will be experienced and dedicated staff offering the same level of professional service. For specialist activities, such as sporting events we will bring in team members that are dedicated to the type of event.
Choose us for your event!
We approach each job with dedicated professionalism, but also a great sense of fun and enjoyment in what we do. We depend on your trust in us to cover your event and reward that trust with exceptional results. We can cater our services to any event, large or small, with the flexibility to provide you with exactly the photography you need..


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Ki-Be Wrestling Home
|Wrestler of the Year
Jesse DeLaPena -152
|Jesse had a terrific year and finished it off with a 4th place finish at state. Among his accomplishments in his career were 82 wins which rank 6th on the Ki-Be all-time list. His 24 post-season wins ranks 7th. He also broke into the top ten in pins, team points and matches wrestled. Jesse made three trips to the Mat Classic and medaled twice. It has been a pleasure to watch Jesse improve as a wrestler over the past four years.
|Wrestler of the Week
|2/4 to 2/10
Sergio Avila
Jesse DeLaPena
Josh Corriell
Eliseo Llerenas
|Ki-Be will be taking four wrestlers to state this year. All four have a good chance of placing. Sergio and Jesse are returning place winners and both will be looking to move up a notch or two. Corriell and Llerenas are newcomers to state and will try not to be awed by the Mat Classic atmosphere.
|1/29 to 2/3 - Justin Corke - 145
|In his first year of highschool wrestling, Justin is going to regionals. If things go well at regionals, he could be going to state. Justin is 13-7 this year and shows an aggressiveness and desire to win. Like his brother Josh, Justin never stops and goes after his opponent for 6 minutes. Justin will be fun to watch for two more years.
|1/22 to 1/28 - Cody Roos - 189
|Cody was 2-0 on the week with a 21 second pin and a forfeit. Cody is a junior and is 13-8 on the year and is tied for the team lead with 9 pins. Cody qualified for regionals last year and is looking for bigger things this year. He will be one to watch in the postseason. He should also have a big senior year.
|1/15 to 1/21 - Kyle Nickerson - 160
|Kyle is the most improved wrestler on the team. After winning just 5 matches in his freshman and sophomore years, he has turned things around and is 14-12 this year. Kyle is a great competitor and does not seem easily intimidated. Kyle has a good chance of placing at District and going to Regionals. The going for state could get tough, but Kyle could rise to the occasion.
|1/8 to 1/14 - Eliseo Llerenas - 215/275
|Eliseo has been a pleasent surprise for the Bears this year. Eliseo was 3-1 on the week, including a third place finish at the Grandview Classic. He has shown the ability to compete with some top notch wrestlers and is 7-2 on the year. Eliseo could surprise all the way to state. The only thing he seems to be lacking is experience.
|1/1 to 1/7 - Josh Corriel - 171
|Corriel had a great week with a pin in less then 1 minute against Wapato. He finished the week with a 1st place finish at the Tonasket Apple Pie Invitational. Josh has been wrestling top notch competition since he started his high school career and he has gained a great deal of confidence and experience. Look for Josh to make a lot of noise come tournament time.
|12/24 to 12/31- Josh Corke -140
|Josh is becoming one of the elite wrestlers in the state. His Royal Invite Championship came at the expense of two wrestlers who defeated him last year. Danny Villarreal of Othello finished third and Russ Chipman of Cashmere finished 4th. Josh defeated Andrew Lynes 8-3 for the title. Lynes finished 3rd place at 1A/B State in 2000.
|12/16 to 12/23- Jesse DeLaPena -152
|After finishing 2nd at the Ki-Be Invite the last two years, Jesse finally broke through with a championship. He recorded two pins on the way to the title which he won on a forfeit. Jesse is currently 7-1 on the year and has won 60 career matches. He should move into the top ten in career wins before the year is over.
|12/8 to 12/15 - Sergio Avila - 119
|Sergio defeated Brad Prather of Connell 7-1 to win the Connell Invite championship at 119 lbs. It was Sergio's 7th career tournament championship. He is now 5-1 on the season and 57-18 lifetime. Sergio has placed 7th and 5th at state the past two years and will be trying to be only the second wrestler in Ki-Be history to place at state in his first three years.
|11/30 to 12/7 - Kevin Hunter - 275
|Kevin recorded two pins during the week against Connell and Othello. He already has improved on his 1-13 record from last year and is gaining confidence with each match he wins. Kevin is aggressive and quick, two things that any heavyweight needs to be competitive. He also has the size to match up well with his CWAC competition. Look for more good things to come from Kevin Hunter this year.


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Would you like to receive sponsorship assistance with an event or program?
To see if your community qualifies, please read the following.
Policy and Procedures for Marketing Expenditures for Economic Development & Community Services
It shall be the policy of the Authority to fulfill the above objectives by establishing the following provisions:
A. GRDA supports and assists activities, organizations, and causes which advance both governmental public purposes, as well as corporate purpose of GRDA by promoting community and economic development, tourism and recreational activities, and conservation and development of natural resources in the communities and industrial areas it serves. This support and assistance will be in compliance with state laws governing GRDA's activities including marketing, consumer education, community relations and customer service functions which it performs.
1. GRDA is a body corporate and politic created by statute and is an agency of the state of Oklahoma. As a result thereof, all funds generated, received and expended by GRDA are public funds and subject to state laws and regulations governing the receipt and expenditure of public funds in the same manner as all other state agencies. Among other activities prescribed by law, GRDA produces, sells and distributes electricity throughout its statutory district, as well as other areas. It is in competition with other electric utilities in the provision of these services.
2. GRDA has had a longstanding practice of supporting and assisting organizations which promote certain public purposes as described above in GRDA's Policy Statement. This practice has been deemed by the Board of Directors of GRDA to enhance its competitive position and to be important generally in carrying out the prudent operation of the business of GRDA.
3. The Board of Directors historically viewed requests for support and assistance in light of whether such a request furthers a public purpose, as well as the corporate purposes of GRDA. The Board finds it necessary and desirable and in the best interest of GRDA and its ratepayers to affirm the application of the public purpose/corporate purpose standard and to better define the working framework for the handling of requests for support and assistance.
C. GUIDELINES FOR GRDA's SUPPORT
1. GRDA may support the following organizational types:
- Community and Economic Development Organizations
- Chambers of Commerce
- Tourism and Recreational Organizations
- Agricultural Organizations
- Environmental Organizations
- Municipals
2. GRDA may support and assist communities that are either served by GRDA, or in which any of its facilities are located, and the GRDA lakes.
3. A budget per community/region will be set based on what is in the best interest of GRDA. Factors taken into consideration will include the number of citizens benefiting from the programs/projects, revenue to GRDA, and the overall need of the community. Any support and assistance provided by GRDA shall be at its sole discretion, provided however, that such support and assistance shall be limited to an amount not to exceed a predetermined total, per year (based on economic conditions) for one (1) or more projects or efforts that are for the benefit of or impact the quality of life for each city or community which GRDA serves or in which it has facilities.
4. GRDA will not monetarily support the following:
- Individuals
- Political campaigns/parties
- Religious organizations
- Private Schools
- Private for-profit organizations
- Solicitations received by form letters
- Groups that discriminate on the basis of age, race, sex, or national origin
- Activities, organizations or causes which do not advance a public purpose and a GRDA corporate purpose
If your community event meets the proceeding criteria, download the request form:
Mail the completed form to:
Lake Relations
PO Box 70, Langley, OK 74350
or fax to 918-256-0906
For more information contact:
Ed Ferguson
Mail the completed form to:
Laura Townsend
PO Box 409, Vinita, OK 74301-0409
or fax to 918-256-5289
For more information contact:
Laura Townsend
Manager Marketing Programs/Community Relations


text:
http://ietf.cse.ucsc.edu:8080/bugzilla/show_bug.cgi?id=145 email@example.com changed: What |Removed |Added ---------------------------------------------------------------------------- firstname.lastname@example.comfirstname.lastname@example.com ------- Additional Comments From email@example.com 2005-12-10 09:46 ------- I think draft 08 is very clear about this (<http://greenbytes.de/tech/webdav/draft-ietf-webdav-rfc2518bis-08.html#rfc.section.14.8.p.4>): "The lockdiscovery property returns a listing of who has a lock, what type of lock he has, the timeout type and the time remaining on the timeout, and the associated lock token. If there are no locks, but the server supports locks, the property will be present but contain zero 'activelock' elements. If there is one or more lock, an 'activelock' element appears for each lock on the resource." So I recommend closing the issue. ------- You are receiving this mail because: ------- You are the QA contact for the bug, or are watching the QA contact.Received on Saturday, 10 December 2005 17:46:46 UTC
This archive was generated by hypermail 2.3.1 : Wednesday, 7 January 2015 15:01:34 UTC


text:
By Diane Bader
I am woman, a seeker after truth, knowledge and Wisdom
One who travels secretly, inwardly, on this journey of life,
One who seeks to know the Feminine,
Who seeks to know the Divine.
I hunger and thirst for inner truth
As I read voraciously,
As I feed upon the wisdom and knowledge of others
Always seeking nourishment for guidance along my own
path of discovery.
When I find such words,
I feast hungrily upon them
Until I find another who adds to the feast
And yet another …
I am refreshed by the sweet nectar
As similar thoughts flow together
To resonate with my soul.
As years flow seamlessly into years,
The urgency of living lessens.
It is my time to grow ever more deeply and
Meditate upon the feast spread before me.
I choose the choice bits
And make them my own
No longer "out there" – someone else's thoughts
They are my soul food.
They belong to me.
Rarely do I find the words to share them,
Rarely do I find an Anam Cara to hear them.
My soul yearns and pines
For the courts of the Divine.
Only then will the feast be complete,
Only then will my soul be satiated.
Meanwhile, I journey upon Mother Earth
Seeking to nourish her and preserve her
For future generations.
I trod gently, very gently
As I enjoy glimpses of the Divine presence
In her sunsets, her gentle rains,
Her green blades of spring,
Her richness of harvest.
When I see brilliant vermilion and orange
Spread across the evening horizon,
I know they are for someone special
Who has just passed from this temporary dwelling
Into the next
And I am again enriched
By the abundance of such Divine Beauty.
* ***
Donna Henes is the author of The Queen of My Self: Stepping into Sovereignty in Midlife. She offers counseling and upbeat, practical and ceremonial guidance for individual women and groups who want to enjoy the fruits of an enriching, influential, purposeful, passionate, and powerful maturity. Consult the MIDLIFE MIDWIFE™
The Queen welcomes questions concerning all issues of interest to women in their mature years. Send your inquiries to firstname.lastname@example.com.
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Product Description
Nike Kyrie 3
Color: Aqua/Black
Style Code: 852395-401
Release Date: August 19, 2017
Dressed in aqua throughout with matching branding located on the tongues and swoosh logos on the sides. The only contrasting detail is seen on the rubber outsole with hits of black to complete the look altogether.
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Authorities and private sector initiatives to increase the number of obtainable jobs and the quantity and size of companies in Indiana have succeeded in the fields of High Tech industries and Life Sciences. Medical and Healthcare jobs are rising and there are increased numbers of jobs available in Excessive Tech industries. We're all crooks and thieves of course! Persons are terrified of what they don't understand. Their car disappears into the building, after a way-too-lengthy of a time frame goes by, they're compelled to empty their pockets. I might love nothing extra then to clarify and educate a customer about the operation of the evaporative emission management system and why it's at the moment not working of their car, however nobody desires to hear any of that. We're additionally not salesman.
Utilizing an company to undergo the hiring course of begins when you submit your resume and job necessities to the company. Prospective employers will interview you both in person or through video call. After you had been authorised, the company will course of your paperwork and requirements with the POEA and in Dubai. They may organize for your visa, working permit, plane tickets, and employment contract. You'll have to pay a placement fee, which is usually equivalent to one month's wage or less.
One factor concerning the cultural festivities and dress of Africans of South Africa, this contains Lesotho, Botswana and Swaziland. These cultural societies have their brand of culture represented fully in South Africa. So that, like the Swazi festivities of the Reeds, the traditional costume of the ladies is a part of the showcasing of the culture-you additionally discover this amongst the Zulus. This about the cultural gown of women is seen in another way by totally different people, globally, once posted on the Web.
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Item Details
Antiques, Tools, Coins, & Accessories
March 10, 2007
Lot #36
Temp #01588
Sold For $90.00 (hammer price)
$103.50 (price including buyer's premium)
Last Updated March 10, 2007
TWO STANLEY BEVEL SQUARES including a #25 Bevel square with a 10" blade and a #24 Patent combination tri-square and bevel with a Shelley's 1864 patent date and a 4" slotted blade.
ConditionIn fine condition.
The description of the item above may not be the final version used in the catalog. The descriptions are as yet unedited and may contain mistakes. The description, estimates and photos could all be wrong. In rare cases they may not end up in this sale. Once the items have received their lot number the descriptions will have been changed if needed.
Hosting • Design • Development: Community Internet Cape Cod SEO


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Funeral for civilians who died in suspected NATO airstrike in Afghanistan
Oct. 5 - Locals in Nangarhar province in Afghanistan hold a funeral for five civilians they say died in an apparent NATO strike on Friday, NATO says unaware of any civilian causalities. Deborah Lutterbeck reports.
Funeral services in Jalalabad for five civilians killed in what Afghan officials say was an air strike by international forces the previous evening. NATO confirmed a strike in the area against insurgents but said initial reports do not indicate that there were any civilian casualties. But some people are still looking for answers. SOUNDBITE: Tribal Elder, Malik Nimat, Saying: "People here are very angry that this incident has taken place and martyred our people. We are asking government officials to talk to the Americans and find out why this incident has taken place." Civilian casualties have long been a source of friction between Afghan President Hamid Karzai and his international backers.


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Organize Your Blog!
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More About Stephanie Giese
Stephanie Giese is the blogger behind Binkies and Briefcases and the founder of the BlogU conference. She has been blogging since 2010, and connecting influencers with brands and hosting large scale events for bloggers since 2014.


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Correction Appended
LOS ANGELES, March 5 - The Academy of Motion Picture Arts and Sciences named "Crash" the best film of the year Sunday, turning its back on "Brokeback Mountain," a film about the love affair between two gay men, which had dominated virtually every other movie award in the run-up to the Oscars.
Ang Lee, however, was the Academy Award for best director for "Brokeback."
"Crash," a film from the director Paul Haggis about a series of tense racial encounters in contemporary Los Angeles, appeared to gain momentum late in the Oscar race, perhaps as a hometown favorite among academy voters.
The academy also honored some of Hollywood's favorite actors as Philip Seymour Hoffman was named best actor for inhabiting the role of Truman Capote and Reese Witherspoon won the best actress award for her performance as June Carter Cash in "Walk the Line."
The 78th Academy Awards, like the year in film, seemed preoccupied with political and moral subjects. George Clooney won the Oscar for best supporting actor for his performance in "Syriana" as a C.I.A. agent caught between terrorists and his corrupt bosses.
And Rachel Weisz was named best supporting actress for her performance in "The Constant Gardener," as a muckraking activist in Kenya whose victimization by an evil pharmaceutical company rouses her emotionally deadened diplomat husband into action.Continue reading the main story
Mr. Clooney, who acknowledged that "we are a little bit out of touch in Hollywood," said that once in a while that was "probably a good thing." And he returned the academy's compliment.
"We were the ones who talked about AIDS when it was just being whispered; we talked about civil rights when it wasn't popular," he said. "I'm proud to be part of Hollywood, proud to be part of the community and proud to be out of touch."
Backstage moments later, Mr. Clooney mused that Hollywood was about two years late getting around to wrestling with the social and political issues confronting the nation. "It won't last very long, but we've done this on and off," he said. "We did it in the 30's, we did it in the 50's, we certainly did it in the 60's and 70's, and we'll probably continue to do that, which is reflect society, not truly lead it."
Mr. Hoffman, accepting his Oscar, congratulated his mother. "We are at the party, Ma," he said. A radiant Ms. Witherspoon, too, thanked her parents. "It didn't matter if I was making a bed or making a movie, they never hesitated to tell me how proud they were of me," she said.
An exception to the dominance of political movies was the winner for best documentary feature, "The March of the Penguins." The surprise hit bested a field that included documentaries on the scandal at Enron and on a hard-fought mayoral election in Newark.
With Jon Stewart, of "The Daily Show," as its host, this Oscars telecast was bound to scald a little, and Mr. Stewart had what were no doubt welcome barbs for Republicans, perhaps less welcome ones for Democrats, and plenty at the movie industry's expense.
He said that the singer Bjork could not attend: "She was trying on her Oscar dress, and Dick Cheney shot her." He suggested that casting their academy ballots may well have been "the first time many of you have ever voted for a winner."
And, in introducing Terrence Howard, who played a pimp in "Hustle & Flow" who aspired to be a rapper, Mr. Stewart defined a pimp as "an agent with a better hat." In fact it was the rap song of Mr. Howard's character, "It's Hard Out Here for a Pimp," by the Memphis group Three 6 Mafia, that won the Oscar for best original song.
Mr. Stewart's narrow and young viewership on the cable network Comedy Central made him relatively unknown to the worldwide Oscars television audience, but his court jester's combination of sarcasm and flattery seemed to work with the Hollywood royalty in the room.
He said Mr. Clooney's "Good Night, and Good Luck" was not just a movie but "how Mr. Clooney ends all his dates." He assured Steven Spielberg that after "Schindler's List" and "Munich," "I think I speak for all Jews when I say, I can't wait to see what happens to us next."
And Mr. Stewart mocked both the show's theme, "A Return to Glamour" -- "For too long Hollywood has done without" -- and the movie industry's obsession with piracy, pointing to its victims in the Kodak Theater. "There are women here who could barely afford enough gown to cover their breasts," he said.
While 2005's movie calendar was short on polarizing events -- no one tackled the Passion or excoriated the Bush administration the way Mel Gibson and Michael Moore had the year before -- it was teeming with modest movies making small statements about Important Subjects.
The nominees for best picture, all but one of them from studio specialty divisions, alone bore evidence of Hollywood's sudden case of seriousness: "Capote" and "Good Night, and Good Luck" cast a critical eye on the roles and responsibilities of journalists, "Crash" explored racial animosity and challenged preconceptions, "Munich" took on Middle East violence, and "Brokeback Mountain" illustrated the point-blank impact of intolerance. The lone big-studio picture, "Munich," was made for some $75 million, more than twice the combined budgets of all four other nominees.
The messages here were seldom radical, the causes rarely unpopular: Niki Caro's "North Country" tackled the evils of sexual harassment, and the point of "Munich" seemed to be little more than the schoolyard lesson that violence solves nothing.
The limits of that seriousness extended, in one direction, to the dumbing down of the Crusades in Ridley Scott's "Kingdom of Heaven," which bombed, and in the other to the fast-paced Mensa test of Stephen Gaghan's "Syriana," whose narrative strands threatened to strangle all but the most attentive viewers.
In a year in which several movies explored terrorism from the perspective of suicide bombers, both "Munich" and "Paradise Now" came under attack for being too kind to terrorists. But the controversy that erupted about "Munich" before it opened dissipated when audiences failed to sit through it in great numbers; in Israel, it received a yawn.
By and large, it was a wretched year for Hollywood's major studios, with a steady stream of action movies and comedies performing belly flops into empty pools.
"King Kong," Peter Jackson's three-hour remake of the epic classic, won the award for best achievement in visual effects, beating out "War of the Worlds" and "The Chronicles of Narnia: The Lion, the Witch and the Wardrobe."
The Oscar for best animated feature went to "Wallace & Gromit in the Curse of the Were-Rabbit," over "Tim Burton's Corpse Bride" and "Howl's Moving Castle."
The costume design award went to Colleen Atwood for "Memoirs of a Geisha," Rob Marshall's wartime Japanese romance. "Narnia" won the award for best makeup.
But few big movies met studio expectations, even those with hopes to contend for best picture: Universal's "Cinderella Man," to name one, won strong reviews but failed to excite mass audiences, while "Memoirs of a Geisha" got neither. And Mr. Jackson's overlong but spectacular "King Kong" never managed the transcendent box office results that made a cultural phenomenon of its director's "Lord of the Rings" cycle.
With none of the best picture contenders reaching huge audiences, and with Harvey Weinstein, late of Miramax, preoccupied with starting a new company and backing only a handful of Oscar contenders, the Oscar campaign season was fairly tame.
About the only thing interrupting the tedium of "for your consideration" ads and breaking the monotony of acclaim for "Brokeback" -- with the exception of the critic Roger Ebert's ardent agitating on behalf of "Crash" -- was an acrimonious lawsuit between the "Crash" producers.
The red carpet offered the usual weave of glamour, fashion and Hollywood striving. Stars who usually move in their own little orbits stood shoulder to shoulder, some hugging, some checking the others with sidelong glances. It can get pretty thick at times, and did. When Mr. Clooney, a nominee in the director, supporting actor and screenwriting categories, reached over to shake the hand of Heath Ledger, a best actor nominee, he did so across Michelle Williams, who had her own nomination as best supporting actress.
Nicole Kidman and Sandra Bullock embraced as Keanu Reeves, who accompanied Ms. Bullock, looked on.
Juicy J, Jordan Houston, a member of the Three 6 Mafia and one of the co-authors of "It's Hard Out Here for a Pimp," stepped up to the Oscars with not one, but two diamond encrusted watches, topped off by a set of diamond chompers, two rows of jewels that he wore over his teeth.
"It's called a grill," he explained. "I got them for some of my music videos, and I just figured, this is the Oscars, I might as well wear them. I'm from hip-hop, and I have to represent by bringing the bling."
Ludracris, the rapper-turned-actor who has a role in "Crash" and was to introduce the performance of Three 6 Mafia, said that Hollywood should get used to it.
"I'm proud to be part of history, the first time hip-hop is performed at the Oscars," he said. Asked whether he was worried how the audience might respond, he said: "I think they will go out an buy the record. It's about time for this kind of music to start showing up."
An outgrowth of the academy's own rules barring more than three producers from receiving the best picture Oscar, a dispute over the film "Crash" pitted Cathy Schulman against Bob Yari, her fellow producer and former partner. In a lawsuit filed even as the last ballots were being cast on Tuesday, Ms. Schulman accused Mr. Yari of acting from "greed and ego" in failing to pay at least $2 million in producing fees to her and her partner, the film's executive producer, Tom Nunan.
Mr. Yari had earlier sued the pair, claiming in January that they had taken funds owed to their joint production company, Bull's Eye Entertainment.
And on Wednesday, Mr. Yari, who put together the $7 million in financing for "Crash," took out full-page advertisements in Hollywood's trade papers with a ringing call to uphold justice and due process by abolishing the secret panels that award credit for best picture.
Academy Award Winners
Best picture: "Crash"
Actor: Philip Seymour Hoffman, "Capote"
Actress: Reese Witherspoon, "Walk the Line"
Supporting actor: George Clooney, "Syriana"
Supporting actress: Rachel Weisz, "The Constant Gardener"
Director: Ang Lee, "Brokeback Mountain"
Foreign film: "Tsotsi," South Africa
Adapted screenplay: Larry McMurtry and Diana Ossana, "Brokeback Mountain"
Original screenplay: Paul Haggis and Bobby Moresco, "Crash"
Animated feature film: "Wallace & Gromit in the Curse of the Were-Rabbit"
Art direction: "Memoirs of a Geisha"
Cinematography: "Memoirs of a Geisha"
Sound mixing: "King Kong"
Sound editing: "King Kong"
Original score: "Brokeback Mountain," Gustavo Santaolalla
Original song: "It's Hard Out Here for a Pimp" from "Hustle & Flow," Jordan Houston, Cedric Coleman and Paul Beauregard
Costumes: "Memoirs of a Geisha"
Documentary feature: "March of the Penguins"
Documentary short subject: "A Note of Triumph: The Golden Age of Norman Corwin"
Film editing: "Crash"
Makeup: "The Chronicles of Narnia: The Lion, the Witch and the Wardrobe"
Animated short:"The Moon and the Son: An Imagined Conversation"
Live action short: "Six Shooter"
Visual effects: "King Kong"
Honorary Award: Robert Altman
Gordon E. Sawyer Award: Gary Demos
Correction: March 10, 2006, Friday An article in The Arts on Monday about the Oscar ceremonies misspelled the name of a rapper who took part. He is Ludacris, not Ludracris.Continue reading the main story


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A Quest Greater Than its Goal
In his introduction to this new edition of the Loeb Classical Library volume Argonautica, editor and translator William H. Race notes that of the three surviving Greek epic poems dated prior to the Roman imperial era, the work of Apollonius Rhodius has not been held in quite as high regard as the other two – namely Homer's Iliad and Odyssey. Citing Longinus' critique of the work, "Apollonius for instance, is an impeccable poet in the Argonautica … Yet would you not rather be Homer than Apollonius?" As Professor Race goes on to note that such words as"inferior," "artificial," "decadent," and "pedantic" have been used by other critics over the past two centuries when considering the writings of Apollonius, it's difficult not to think anyone would not indeed choose to be Homer. Read the rest of this entry »


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The Mundus Group, Inc. (MNDS.PK) is a provider of advanced aerospace technology for civilian and military uses. Through its wholly owned VTOL division, Roadable Aircraft International (RAI), The Mundus Group provides the patented Vertical Take Off and Landing (VTOL) technology for experimental aircraft and Unmanned Air Vehicles (UAV).
The Mundus Group plans to use RAI to develop and market the world's first commercially successful vertical takeoff and landing flying vehicle. The idea is to develop a vehicle with the ability to take off without a runway, be able to land in small spaces, and be able to drive to the final destination. Their first flying car prototype won first place and best invention of the year in 1993 at the Discovery Channel Invention Convention.
The Mundus Group believes that the need for alternative transportation will increase along with the public's awareness for this need. The company is in a good position to emerge as a market leader due to the untapped market for a "flying car", and their existent, advanced research, technologies and prototypes.
Let us hear your thoughts: The Mundus Group, Inc. Message Board


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What is your most recent book? Tell us a bit about it.
My most recent book is Mood Matters: From Rising Skirt Lengths to the Collapse of World Powers, which is an extended account in words and pictures of how the mood of a population (how it feels about the future, optimistic or pessimistic) dramatically impacts the types of collective events we can expect to see. This ranges from short timescale events like the kinds of films people will like and the sorts of fashions that will be trendy to longer-term events like shifts in political ideology to very long-term events such as the demise of a global power or even a civilization.
Tell us something about yourself.
I was born and raised in Portland, OR and received all my university degrees in mathematics. But that was decades ago, and since finishing my studies I've been a university professor and researcher at a number of major international centers?The RAND Corporation, the Santa Fe Institute and the Int'l Institute for Applied Systems Analysis (IIASA) in Vienna, Austria. Currently, I lead a program at IIASA devoted to the study of extreme events in human society.
I started writing general-audience books about twenty years ago, with the best-selling volume Paradigms Lost: Unsolved Mysteries of Modern Science. Since that time I've written a dozen popular-science volumes, including Complexification, The Cambridge Quintet and Would-Be Worlds.
What inspired you to write this book?
I wanted to present an account of the developing science of socionomics to a broad audience. I feel the big idea underlying socionomics, the notion that it is the mood of a group that drives social events, not other events, is crucial to understanding how things like the current financial crisis, presidential elections and trends in popular culture.
How did you choose the title?
By focusing on what, exactly, the book is about (social mood) and why I wrote the book (because it is the mood that matters insofar as why we see what we do and not see something else).
What obstacles did you encounter in getting this book published? How did you overcome them?
The major obstacle was simply that editors, like just about everyone else, simply did not believe the basic premise of the book that it is the social mood, not events, that underlies what we can expect to see.
I cannot really say I "overcame" those obstacles, other than to continue to look for a sympathetic editor who wasn't afraid to take a chance on publishing a far-from-mainstream idea. That took about five years!
How did you know you wanted to be a writer? How did you get started?
Despite the fact that I've written over a dozen general-reader books, I'm not sure I really consider myself a "writer". Mostly, I got started because there were ideas/questions I wanted to know more about (origin of life, the possibility of a thinking machine, the existence of intelligent lifeforms elsewhere in the universe, and so on), and thought that the best way to prove to myself that I understood these questions was to try to explain them in everyday language to someone else.
Do you have any writing rituals?
Not really. I just do a lot of background research, try to assemble it into a coherent story and then start putting down one word after another. Of course, rewriting is the key as my manuscripts undergo many revisions and tweaking before they see the light of day in print.
Did you learn anything from writing and publishing this book? What?
Never give up when everyone says you're wrong; give up immediately when everyone says you're right!
If you were doing it all over again, what would you do differently?
I would start writing general-reader books sooner!
What types of books do you like to read? Who are your favorite authors? Why?
I read many different types of books, ranging from professional reading in computer modeling and system theory to airport thrillers. In this latter category, I'm partial to authors, mostly British it seems, who almost never show up on bestseller lists, such as Peter James (not P.D.!), Philip Kerr, Barry Eisler, Tess Gerritsen, to name but a few. I also like "hard" science fiction, as exemplified in the work of Greg Benford, Robert Sawyer, and more classically-speaking, Stanislaw Lem. I like all these authors because they tell good stories and do very good research. So it's a pleasure to be both educated and entertained in a single sitting.
Are you working on your next book? What can you tell us about it?
My next book will be a general account of extreme events, especially those brought about by human fraility like financial meltdowns, Internet failures, political revolutions, terrorism and the like. The book will address issues such as why and how such events happen, their effect on society, and most importantly, what sorts of tools we can employ to anticipate and, hopefully, head off such events before they fully unfold.
What is the best advice you could give other writers about writing or publishing?
Follow your muse, don't give up when people ignore your work, and most importantly, don't write for money. Do it for self-fulfillment and enjoyment!
Who is the perfect reader for your book?
An intelligent, open-minded layperson (not an academic or even intellectual) who wonders about why collective events happen one way and not another, and how we can understand the forces driving such events. Such a person might be from any walk of life and may have a "day job" of almost any type ranging from a dance instructor to a nuclear physicist. The most important factor is that they have some intellectual curiosity about the world around them and are not afraid of thinking unpopular or even counter-intuitive thoughts.


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Charlene Arje
Executive Advisor, CEO/President Groups, PEO
Charlene brings her extensive experience and positive energy to PEO as an Executive Advisor. She is passionate about leadership, business best practices, teamwork and mentorship.
Charlene has had a successful 30 year business career in the corporate and entrepreneurial sectors. At Honeywell, where she spent 12 years, Charlene moved from a financial leadership role to a leader in corporate integration and shared services. In 1999, she led the integration of AlliedSignal Canada and Honeywell Canada, into a $1 Billion Company. Afterwards, she created the Shared Services Division for Honeywell Canada, bringing together HR, Finance, IT from 20 locations across Canada. From there, she assumed greater responsibility for Honeywell global operations, and ran a number of their Shared Service Organizations. In addition, Charlene was appointed President of Honeywell Canada.
After receiving her Bachelor of Commerce from McGill University, Charlene relocated from Montreal to Toronto, where she did her MBA at York University, and then started her career at Ford Motor Company of Canada. Charlene was at Ford for 15 years, honing her financial skills, in increasingly responsible financial positions.
Charlene is married with children and grandchildren.
- Financial and Business Acumen
- Negotiations
- Leadership, mentoring and team-building
- Analytics, scorecards and metrics – Using data to improve performance


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"The everyday things we do in the classroom are deeply important, but sometimes, because they're often the most mundane, they're overlooked." - Doug Lemov, Teach Like a Champion (Interview with Kid-Smart Spaces)
Hi! My name is Magalie René-Hayes. I've transformed many chaotic, depressing spaces but I've also seen beautiful classrooms arranged by great teachers.
Every child deserves to learn in a space that inspires them. And every TEACHER deserves spaces that support the job they do! Because let's keep it real, doing the world's most important job is no easy feat!
In this book I'll share my design and organization methods, key research on how design affects learning, and most importantly, tips from the teachers who do it best.
YOUR CONTRIBUTIONS will create the opportunity to share this book with teachers across the country and touch the lives of countless students.
The proceeds will go to:
- Transcription Services - to transcribe recordings including several expert interviews: Dale Caldwell, CEO of The Village Charter a charter school in Trenton, NJ on its way to becoming a Blue Ribbon school despite it's location in one of the countries most challenged districts and Doug Lemov, highly respected educator and author of Teach Like a Champion and Reading Reconsidered.
- Editorial Services - 1. Developmental Editor who will evaluate and critique the manuscript, suggest and provide revisions to make sure everything flows and is consistent. 2. Copy Editor to catch spelling mistakes and adjust for grammar, punctuation and consistency. Great editors charge what they're worth. This book deserves great editors.
- Book Design + Layout - The cover design needs to be optimized for print, digital, thumbnail sizes, and designed on an e-readers/ mobile devices because readers judge how a book looks on a shelf and how it looks on an iPad or black-and-white Kindle.
- Printing - Books will mainly be printed on-demand however the goal is to print advanced copies to support marketing efforts including speaking engagements, editorial samples, gift/donations, and reviews.
- Marketing/Publicity - The importance and impact of classroom and school design is still underestimated. A publicist will help me spread the message behind this book far and wide. Because the more we educate and the more widely this information is shared, the more impact we have on policy-makers and on the education system.
- Photographer


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If you've got your hands on the 2015 Stampin' Up! Holiday Catalogue, you'll recognise my project today. This week at CASEing the Catty, we're featuring all the brand new papers in the catalogue. I've always been a fan of papers. Different colours, textures, weights, patterns. I thought there was no better project in the catalogue to showcase papers than the Christmas stars featured on page 22.
This catalogue was 'unveiled' at Stampin' Up!'s US convention a few months ago and one of the wonderful staff demonstrated how to create the stars. It's really very simple.
I started with two 6″x6″ pieces of paper. Fold each of them half (to make a rectangle), unfold. Turn 90 degrees and fold in half again. Unfold. Fold in half diagonally as well from corner to corner, and then repeat for the other corners.
Then cut a small slit 3cm (or 1 1/4″) up each of the four straight edge folds (NOT the diagonal folds). Fold from the slit into the centre of the diagonal fold and repeat for the other side, making a kite shape at each corner. Repeat for all corners. Now apply some glue to one half of the 'kite'. Overlap the second half kite and adhere to the top. Once you've done this for all four corners, you'll have made one half of the star.
Simply repeat this process for the second sheet of paper and adhere them together.
I decided to make three stars in different types of paper.
The first uses the Holidays Fancy Foil Designer Vellum. I love vellum and I love the plaid look the overlapped pieces add to the star as well. Vellum can be difficult to adhere because you can often see the adhesive through it but I find the new Fine-Tip Glue Pen works really well.
The next star features traditional weight designer series paper. I used the retro/vintage Home for Christmas DSP. Let me tell you, this pack is just gorgeous. It was SO hard to decide which sheet to use.
Finally I wanted to try something a little different. I decided to use the lighter weight envelope paper. It has a beautiful texture and I love the way it turned out.
I've really only just scratched the surface with these stars of mine. You could try all sorts of papers! A gold one for the top of your tree? Baby size stars as tree ornaments? Use embossing folders or scoreboard to add texture to plain paper. The possibilities are just about endless. I'd love to see these start popping up all over the place. Don't forget to try them out and join in with the CASEing the Catty Crew. If you Instagram, let me know you've had a go at creating them and tag @thepapercaper!
Next up on the blog hop is the wonderful Alison Barclay. If you've missed someone and need to go back, visit Monique Fielder or head to HQ for a full list of blog hoppers.
Jess x