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The National Institute for Genealogical Studies Announces a New Eastern European Records Certificate
Features Courses Written by Lisa Alzo, MFA
For Immediate Release
27 February 2018
The National Institute for Genealogical Studies is proud to announce a new addition to our Certificate Programs. The Eastern European Records Certificate features courses in researching various records and repositories in Eastern Europe.
Course author and genealogy professional, Lisa Alzo, MFA says of researching Eastern European ancestors, "A vast number of immigrants came to the United States and Canada from various areas in Central and Eastern Europe in the late 19th and early 20th centuries. Unfortunately, in the past, those with Eastern European roots have been reticent to begin research on their immigrant ancestors due to perceived barriers of language, geography, and difficulty with accessing and understanding the records. With the popularity of DNA testing, more people are discovering they have Eastern European ancestry and have a desire to know more. As more records become accessible with each passing year, it has never been easier to consolidate known facts with newly discovered information."
Courses in this new certificate program include:
Other courses offered include research, records, and repositories in individual Eastern European countries and regions.
The certificate in Eastern European Records will provide in-depth instruction for genealogists with roots in many countries where research was difficult, if not impossible, just a decade ago.
"We are excited to announce the creation of this new certificate program focusing on Eastern Europe. Researchers will find these courses to be an important addition to expanding their knowledge about the region and records," remarked National Institute's Managing Director Louise St Denis.
The Eastern European Records Certificate description and links to courses can be found on The National Institute's website at http://www.genealogicalstudies.com/eng/courses.asp?certificateID=25. Courses are continually being added to this certificate program.
In conjunction with the RootsTech conference, The National Institute is offering a 20% discount off any Eastern European course package. To register for a course package, use code 20RT18EE at checkout. Eastern European course packages can be viewed on The National Institute's website at http://www.genealogicalstudies.com/eng/packages.asp.
The National Institute for Genealogical Studies, leaders in online genealogy education, has been offering genealogy and history courses for 20 years. Over 200 courses in genealogical studies are offered to help enhance researcher's skills.
For those looking to acquire more formal educational training, The National Institute offers Certificate Programs in the records of Australia, Canada, England, Germany, Ireland, Eastern Europe and the United States, as well as a General Methodology, Professional Development and Librarianship Certificate Program. For more information please call us toll-free in North America at 1-800-580-0165 or email us at firstname.lastname@example.com.
For more information:
Louise St Denis
1-800-580-0165 (North American)
416-861-0165 (International)
Skype: louisestd
Last updated by Gena Philibert Ortega Feb 27, 2018.
© 2019 Created by Nat Ins for Genealogical Studies. Powered by


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Amazing Encounter – Sea Kayaking with Orcas (Video)
What an Experience!
These are our taonga, our treasures, and we MUST take better care of the oceans, and not for us, but for those creatures that call the ocean their 'home'.
Nathan Pettigrew
Nathan Pettigrew from New Zealand shot this terrific footage when sea kayaking from Pilot Bay to Mauao (Mount Maunganui).
He caught sight of 2 orca going passed him in the water and stopped to find the rest of the pod appearing in front of his kayak.
The whole pod were happily feeding on eagle rays while Nathan just sat back and filmed this amazing encounter.
Nathan, like many of us wants to help protect our oceans and the beautiful creatures that live there and if we all do our bit to help with conservation then sea kayaking encounters like these will be here for generations to come.
Enjoy the grace and beauty of these amazing orcas in the video on the next page . . .

subset: commoncrawl


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iJoy Avenger 270 234W Voice Control Kit
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All pictures are based on practicality, copyright reserved.


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Budget smartphone Meizu M5C will appear in Russia in June this year
The company Meizu announced a new smartphone Meizu M5C, which belongs to the budget class. The smartphone has a polycarbonate shell and five color options.
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The price is not reported, but it is known that the device will appear in a number of countries, including Russia and Ukraine, in June.


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Cytochrome P450 associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae
Contributed by Janet Hemingway, June 14, 2016 (sent for review February 26, 2016); reviewed by Jeffrey Bloomquist, Basil D. Brooke, and Pie Mueller
Malaria incidence has halved since 2000, with 80% of the reduction attributable to the use of insecticides, which now are under threat of resistance. Understanding the mechanisms of insecticide resistance is a key step in delaying and tackling the phenomenon. This study provides evidence of a cuticular mechanism that slows the uptake of pyrethroids, contributing to the resistance phenotype and potentially broadening resistance to multiple insecticide classes, thus providing additional challenges to resistance management. Quantitative modification of cuticular hydrocarbons is associated with increased expression of a 4G cytochrome P450 enzyme, CYP4G16, which catalyzes epicuticular hydrocarbon biosynthesis. This work improves our understanding of insecticide resistance and may facilitate the development of insecticides with greater specificity to mosquitoes and greater potency.
The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of 14C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An. gambiae.
Pyrethroid resistance in malaria vectors of human disease is at a critical tipping point. Resistance is now widespread and is rapidly increasing in intensity in Anopheles mosquitoes across Africa (1, 2). A key challenge is to maintain the efficacy of current interventions in the face of growing insecticide resistance. Overexpression of detoxification enzymes, which inactivate or sequester insecticides, and mutations in the target site that alter the affinity of insecticide binding have been widely described in the major malaria vector Anopheles gambiae (3). However, the emergence of striking multiple-resistant phenotypes in West Africa, where mosquito populations with very high pyrethroid resistance levels are also resistant to additional classes of insecticides (4⇓–6), suggests the emergence of additional broad-spectrum mechanisms.
The cuticle has been hypothesized to play a role in insecticide resistance via modifications that reduce or slow insecticide uptake. The phenomenon has been studied in some agricultural pests (7⇓–9). Cuticle thickening, hydrocarbon (HC) content, and reduced deltamethrin penetration have been associated with high levels of pyrethroid resistance in the hemipteran Triatoma infestans, the vector of Chagas disease in the Americas (10, 11), and cuticular resistance has been demonstrated in a dichloro-diphenyl-trichloroethane (DDT)-resistant Drosophila strain (12). Cuticular resistance has been hypothesized in mosquitoes, but empirical data are confined to a report that increased cuticle thickness is associated with pyrethroid resistance in Anopheles funestus (13). Transcriptomic studies show substantial overexpression of multiple cuticular genes in several resistant Anopheles and Aedes mosquito populations (14⇓–16). Genes possibly involved in the biosynthesis of lipid components and fatty acid metabolism are also associated with resistant phenotypes in Anopheles arabiensis and An. gambiae (16, 17). In particular, the cytochrome P450 cyp4g16 and cyp4g17 genes are overexpressed in resistant populations of both An. gambiae (18) and An. arabiensis (17) in several regions in Africa.
Unlike the Anopheles P450 cytochromes agCYP6M2 and agCYP6P3, which are localized primarily in Malpighian tubules and the mosquito gut (19, 20) and metabolize pyrethroids, no role in insecticide resistance has been attributed to CYP4G17 and CYP4G16.
These P450 cytochromes are the only known members of the insect-specific 4G family of P450 cytochromes in An. gambiae. They are potential homologs of Drosophila dmCYP4G15 and dmCYP4G1. The latter has decarbonylase activity involved in the final step of cuticular hydrocarbon (CHC) biosynthesis from aldehyde precursors (21). In Drosophila dmCYP4G1 is localized in the CHC-secreting oenocytes in the abdomen, and dmCYP4G15 transcripts are enriched in the head and nervous system (21, 22). Both An. gambiae cyp4g17 and cyp4g16 transcripts are highly enriched in the abdominal integument (18).
Here we analyzed barriers to insecticide uptake through the cuticle by comparing the rate of internalization of radiolabeled pyrethroid and the response to different modes of insecticide application in resistant and sensitive strains of mosquitoes. We subsequently investigated possible cuticular modifications between resistant and susceptible mosquitoes by transmission electron microscopy (TEM) and quantitative GC/MS approaches. Finally, we analyzed the expression profile, subcellular localization, and activity of the Anopheles 4G P450 cytochromes to investigate their function in the CHC pathway and possible role in resistance.
Insecticide Uptake Is Slower in Resistant Insects, and Resistance Is Partially By-Passed by Acetone/Topical Application.
Barriers to uptake were monitored initially by comparing the rate of internalization of a WHO standard oil-based formulation of radiolabeled insecticide after (tarsal) exposure on filter papers in the resistant and sensitive strains. By comparing radiolabeled 14C extracted from the cuticle with residual 14C in the whole mosquito, we observed that the internalization of deltamethrin was ∼50% slower in resistant than in susceptible mosquitoes (Table 1). In addition, there was a 50% drop in the resistance ratio (from 22.4-fold to 12-fold) when the insecticide was applied topically in acetone as compared with the contact-exposure assay (Table 1).
TEM Analysis of Cuticle Thickness.
To overcome issues in comparisons within and between samples, the apical region of the femur leg segment was analyzed. The same number of sections was taken in both samples, and measurements in each section (>10) were done randomly (Fig. 1A). The overall cuticle thickness was variable across legs but was significantly higher in the resistant strain (2.13 ± 0.32 µm) than in the sensitive strain (1.873 ± 0.30 µm) (P < 0.05) (Fig. 1B, Left). The thickness of epicuticle was much less variable and was significantly higher in the femur from resistant mosquitoes (resistant epicuticle = 0.57 µm ± 0.11 µm vs. susceptible epicuticle = 0.27 ± 0.04 µm; P < 0.01) (Fig. 1B, Right) and thus was the major contributor in the overall difference in the total thickness of the cuticle.
Resistant An. gambiae Mosquitoes Contain Larger Amounts of CHC than Susceptible Specimens.
TLC analysis indicated that HCs were the most abundant components in the lipid layer of mosquito cuticle (Fig. 2A, Inset). GC analysis of CHCs and total mosquito HCs showed indistinguishable HC profiles by both GC-flame ionization detector (FID) and GC-MS (Fig. 2A), with CHCs representing 85.38 ± 0.99% of total HCs in resistant females and 81.91 ± 2.84% in susceptible females. A representative HC profile of adult An. gambiae is shown in Fig. 2A (also see Dataset S1). A previous report described 48 CHCs in Anopheles coluzzii, including n-alkanes, monomethyl, single-component dimethylalkanes, and small amounts of alkenes (23). Here, we extend this identification to 70 multicomponent peaks and identify a variety of multi-isomeric methyl- and dimethyl-branched components (Dataset S1). Saturated very-long-chain dimethyl- and monomethyl-branched components were the predominant structures. The relative abundance of multiple isomers of dimethyl-branched high-molecular-weight components, mostly of 39–47 odd-numbered carbon backbone, is remarkable (accounting for >50% of the HC blend). A multiplicity of 13,x-, 11,x-, and 15,x- isomers was found in most peaks, with x ranging mostly from 17 to 33 (Fig. 2A and Dataset S1), followed by a series of internally branched monomethylalkanes up to a C47 backbone, minor amounts of even-numbered dimethyl- and monomethyl-branched chains, and odd-numbered straight-chain components from nC23 to nC33. Small amounts of straight-chain monounsaturated components up to 47 carbons, with double bonds located mostly in positions 9-, 7-, and other internal locations, were also identified.
Subsequently, CHCs derived from the resistant and the susceptible strains were compared. GC-FID analyses showed that, although there were no qualitative differences in the CHC profiles of resistant and susceptible female mosquitoes, the resistant mosquitoes contain significantly higher amounts (∼29%) of CHCs than susceptible specimens (Fig. 2B).
Association of the 4G Family of P450 Cytochomes with the CHC Production Pathway.
The finding of higher levels of CHCs in resistant mosquitoes supported a previous hypothesis that the increased expression of Anopheles 4G P450 cytochromes in resistant mosquitoes might be associated with up-regulation of the HC production pathway. To examine the function of the two Anopheles 4G P450 cytochromes found in the Anopheles genome, we undertook a series of experiments to identify cyp4g transcripts (Fig. S1), together with characterization of the subcellular localization and decarbonylase activity of the proteins encoded.
CYP4G17 Is Dispersed Throughout Oenocyte Cytoplasm, Whereas CYP4G16 Is Associated with the Intracellular Side of the Plasma Membrane.
Longitudinal sections from frozen prefixed mosquito specimens were immunostained with α-CYP4G17–, α-CYP4G16–, and α-CPR–specific antibodies (green in Fig. 3A, Left). Both CYP4G16 and CYP4G17 gave very strong signals and thus were likely to be highly abundant in oenocytes (Fig. 3A), a cell type thought to secrete HCs. This group of cells, with a characteristic lobe shape, has previously been shown to express high levels of cytochrome P450 reductase (CPR), the obligatory P450 electron donor (24). We were unable to detect specific signals in other tissues by immunostaining.
Surprisingly, high-magnification confocal microscopy focusing on oenocytes revealed different subcellular distributions for CYP4G17 and CYP4G16 (Fig. 3B); CYP4G17 resides throughout the intracellular region, similar to CPR, as expected from a microsomal P450 protein, whereas CYP4G16 is found at the periphery of the oenocyte cell, presumably associated with the plasma membrane (PM). The specificity of the antibody was checked by silencing the corresponding gene, followed by immunofluorescence (Fig. S2A), and further immunofluorescence experiments in the presence or absence of detergent (± Triton X-100) (25) demonstrated that CYP4G16 is associated with the PM intracellularly (Fig. S2B).
CYP4G16 Encodes a Functional Oxidative Decarbonylase.
To provide direct evidence of HC synthesis involvement through an in vitro decarbonylase assay, CYP4G16 and CYP4G17 were expressed as fusion proteins with CPR (from housefly) in Sf9 (insect) cells using a baculovirus expression system similar to that described by Qiu et al. (21). As shown in Fig. 4A, the CYP4G16 fusion preparation converted C18 aldehyde to HC with efficiency similar to that of the housefly CYP4G2 protein and showed time-dependent decarbonylase activity with this substrate (Fig. 4B). A similar fusion protein, CYP4G17, was produced at a lower concentration than CYP4G16 and did not demonstrate aldehyde decarbonylase activity with the C18 aldehyde substrate (Fig. 4A). Longer aldehydes could not be tested because of solubility and the unavailability of labeled substrate.
The reduced penetration rate of 14C deltamethrin in a highly resistant strain of An. gambiae suggests that cuticular modification may affect insecticide uptake. Similarly, reduced deltamethrin penetration was observed in a T. infestans resistant strain (10), in which removal of the epicuticle lipid layer with solvents correlated with enhanced insecticide penetration and insect mortality (26). A slower rate of penetration into the mosquito may enhance resistance by increasing the time available for metabolic processes to inactivate the insecticide before it reaches the target site. Possible cuticular modifications might include changes in the thickness of the cuticle or qualitative modification of its components. In the strains examined, resistant mosquitoes had a thicker epicuticle layer than susceptible mosquitoes and contained greater quantities of CHCs, but qualitative CHC profiles were not altered. The possibility that differences in the size of individuals or in their genetic background might introduce variability in cuticle thickness measurements cannot be excluded, although the average weight of the resistant and the susceptible mosquitoes was not different (resistant = 1.07 ± 0.118 mg; susceptible = 1.14 ± 0.102 mg, n = ∼500), and both strains were of the same molecular form and originated from West Africa.
Interestingly, adult An. gambiae have a considerably higher CHC/weight ratio (∼300–400 ng/mg) than reported for T. infestans (30–40 ng/mg) (11). In addition, in An. gambiae CHCs represent the majority (>80%) of the total mosquito HCs, with a composition similar to that of the internal pool. In insects such as triatomines and cockroaches, only ∼10–20% of the HCs are transported to the surface, and the majority of the methyl-branched HCs are retained in the hemolymph as reservoir (27, 28).
In addition, the An. gambiae strains examined have a distinct and remarkable abundance of very-long-chain methyl-branched HCs (VLCMHCs) compared with other insects examined to date (29). VLCMHCs have been associated with increased waterproofing properties (30) and may be correlated with a need for greater desiccation resistance in these relatively small insects.
Quantitative GC/MS on whole-body extracts from pools of mosquitoes showed that resistant mosquitoes contain more CHCs than susceptible strains, as had been shown in similar studies with T. infestans (10, 11). We hypothesized that increased HC production in resistant Anopheles might be associated with increased expression of CYP4G16 and/or CYP4G17 and might contribute to resistance by changing the permeability and insecticide-uptake rate (16). To investigate the potential function of the two 4G P450 cytochromes, we defined their localization and in vitro oxidative decarbonylase activity.
Previous transcriptomic analysis of dissected mosquitoes indicated high levels of cyp4g16 and cyp4g17 mRNA in the abdominal integument (18). We now show that within this abdominal region the respective proteins are highly abundant in oenocyte cells, which are known to produce and secrete HCs in other insects (21, 31, 32). The two P450 cytochromes have different subcellular localizations: CYP4G17 was found throughout the intracellular compartment, as expected for a general endoplasmic reticulum (ER)-bound P450 protein (similar to CPR), and CYP4G16 was predominantly associated with the cytoplasmic side of the cell membrane. We cannot be sure whether this localization is within the PM or is part of the distal ER that may associate closely with the PM (33). This site appears to be unique for a P450. A small number of mammalian P450 cytochromes have been localized within PMs. However, in these cases the major location site remains on the ER (34). Furthermore, these mammalian membrane-bound P450 cytochromes have catalytic surfaces facing the extracellular space (35, 36).
The different localization of the two P450 cytochromes may indicate differences in CYP4G16 and CYP4G17 cellular functions. One possible scenario could be that the proteins display different substrate specificities. So far, only CYP4G16 has been found to have in vitro decarbonylase activity against a C18 aldehyde substrate to produce HCs. Our efforts characterize the function of CYP4G17 fully were inconclusive, mainly because the protein was not stably expressed. However, we cannot rule out the possibility that CYP4G17 (and CYP4G16) can catalyze the conversion of lipids of different molecular weights and/or carrying methyl-branched precursors.
Given its putative role in epicuticular lipid biosynthesis, the finding that Anopheles CYP4G16 protein exerts its decarbonylase activity associated with the PM is intriguing. The biosynthetic machinery responsible for lipid metabolism and the production of CHCs in other insects is thought to be localized on the smooth ER (37, 38), although there are exceptions (39). Further studies are needed to establish the localization of the other CHC enzyme machinery in An. gambiae, including elongases, desaturases, and reductases that would be expected to reside in the oenocytes. Our working hypothesis is that newly synthesized, very hydrophobic CHCs could be manufactured and loaded directly onto lipophorin at the lipophilic interface of the cell membrane for transport to the cuticle (32, 40). In addition to the biosynthetic enzymes, an electron donor, such as the ER-bound CPR, is necessary for P450 activity. These studies indicate a degree of positional overlap of CPR with CYP4G16, indicating that CPR may well retain its function as an electron donor with P450 cytochromes in this PM-associated location (Fig. S2C).
A functional in vivo link between the CYP4G16 and the synthesis of epicuticular lipids was not observed following cyp4g16 RNAi silencing. Although diminishing the oenocyte-expressed protein was successful at the adult stage (Fig. S2A), no significant differences were observed in CHC content or composition. One possibility is that CHC biosynthesis is less active in adult oenocytes, and large deposits of CHC are generated during the late larval and pupal stages. The application of RNAi at earlier (pupal or larval) stages was not feasible, and it is likely that a transgenic approach to manipulate CYP4G16 and CYP4G17, specifically at earlier mosquito stages, is needed to elucidate this issue.
Together the data suggest a role of CYP4G16 in insecticide resistance via enrichment of the CHC content, thus reducing pyrethroid uptake. The cuticular-based resistance mechanism, in addition to targeting site resistance and detoxification, further strengthens the insecticide resistance phenotype and potentially broadens it to multiple insecticide classes, thus providing additional challenges to resistance management. The Tiassale strain used in this study is one of the most highly resistant field strains of An. gambiae, with resistance encompassing all currently used classes of public health insecticides (4). Selection for and spread of such broad-spectrum mechanisms should be avoided; otherwise, resistance to insecticides could have a massive impact on our ability to prevent malaria and other vector-borne diseases.
Materials and Methods
Mosquito Strains and Bioassays.
The An. gambiae strain N'Gusso, from Cameroon, is susceptible to all classes of insecticide. The Tiassale strain, from southern Cote d'Ivoire, is resistant to all insecticide classes (4). Mosquitoes were reared under standard insectary conditions at 27 °C and 70–80% humidity under a 12-h:12-h photoperiod. Bioassays, including tarsal contact and topical application of the insecticide deltamethrin, were conducted as previously described (41), with minor modifications. For tarsal contact exposure, 10–13 mosquitoes were exposed to the coated surface of a Petri dish. For topical application, mosquitoes were briefly anesthetized using CO2, and 0.25 μL of insecticide solubilized in acetone was carefully applied to the dorsal thorax using a Burkard hand microapplicator (Burkard Scientific). Five replicates of 10 mosquitoes were exposed to each insecticide concentration. After exposure, mosquitoes were maintained at 27 ± 2 °C and 70 ± 5% relative humidity with access to a 10% (wt/vol) sucrose solution. Mortality was assessed 24 h after exposure.
Penetration Rate of 14C Deltamethrin.
Thirty to thirty-five female mosquitoes from each strain were exposed for 3 min to radiolabeled 0.01% deltamethrin, donated by Ralf Nauen, Bayer CropScience, Monheim am Rhein, Germany, on impregnated filter paper, using standard WHO bioassay protocols (apps.who.int/iris/bitstream/10665/64879/1/WHO_CDS_CPC_MAL_98.12.pdf). At the end of the exposure, mosquitoes were collected and rinsed three times with 1 mL hexane. The washes were retained. Mosquitoes were allowed to dry before homogenization in 500 μL PBS. Ten milliliters of liquid Scintillation Counting Mixture (Ultima Gold; 6013326; PerkinElmer) was added to each sample, and the corresponding counts per minute were measured on a beta counter (LS1701; Beckman). The penetration rate (PR) was calculated as the ratio of internal counts per minute to total counts per minute.
The apical regions of the femur leg segment of 25 susceptible and 25 resistant female mosquitoes were dissected and fixed in 2% (vol/vol) glutaraldehyde, 2% (vol/vol) paraformaldehyde in 0.1 M sodium cacodylate buffer (SCB), pH 7.4, for 24 h at 4 °C with constant rotation. The samples were washed three times for 5 min each in 0.1 M SCB, postfixed in 1% aqueous OsO4 for 12 h at room temperature with constant rotation, and then washed again as above. After the last washing, the samples were stained with 2% (vol/vol) uranil acetate for 1 h. Then the samples were rinsed with SCB, dehydrated through an ascending acetone gradient [30–50–70–90–100% (vol/vol)], infiltrated with Durcupan ACM Fluka resin [3:1 propylene oxide:resin mixture for 1 h followed by a 1:1 and a 1:3 propylene oxide:resin mixture for 1 h each and finally 100% (vol/vol) resin for 16 h], and embedded in flat molds. The resin was cured in a drying oven at 60 °C for 48 h. Samples were trimmed, thin-sectioned (gold section), and absorbed onto 300-mesh copper grids. Observation was carried out using a high-resolution JEM 2-100 transmission electron microscope (JEOL) at an operating voltage of 80 kV.
Extraction of Lipids, Separation by TLC, HC Fractionation, and Derivatization.
Cuticular lipids from female mosquito specimens were extracted by three 1-min immersions in hexane with gentle agitation, and extracts were pooled and evaporated under a N2 stream (20 insects per tube, n = 5 resistant and n = 4 susceptible). Internal HCs were further extracted from the same samples after 24-h immersion in 2.5 mL hexane and a final hexane washing (2 mL). Total HCs were extracted in the same way without prior extraction of cuticular lipids. The lipid extracts were analyzed by TLC on 4 × 8 cm Polygram Sil G/UV254 silica gel plates (Macherey-Nagel) using two development solvent mixtures: n-hexane [100% (vol/vol)], followed by n-hexane:ethyl ether:acetic acid [80:20:1 (vol/vol)]. Plates were sprayed with 5% sulfuric acid in 95% (vol/vol) ethanol, and lipid bands were visualized after charring at 180–200 °C for 20 min. HCs were separated from other components by adsorption chromatography on a minicolumn (2.5 × 0.5 cm i.d.) of activated SUPELCOSIL A (Supelco), eluted with hexane (4 mL), and then concentrated under an N2 atmosphere. Double-bond location was detected after derivatization of the HCs according to Carlson et al. (42). Total HC extracts obtained from 100 susceptible female mosquitoes were solved in hexane (100 μL) and treated with 100 μL of dimethyl disulfide and 50 µL of iodine solution in diethyl ether (60 mg/mL). The reaction mixture was held for 4 h at 40 °C, diluted with 0.5 mL of hexane, and treated with Na2S2O3 10% (wt/vol) (Merck) until the iodine color disappeared. The organic phase then was separated and evaporated under N2.
CHC Quantitation and Identification by GC and GC/MS.
GC analyses were performed using a Hewlett-Packard 6890 gas chromatograph with an FID and equipped with a split/splitless injector port, fitted with a high-temperature capillary column (30 m L × 0.25 mm i.d., 0.25 μm film thickness) (Zebron ZB-5HT Inferno; Phenomenex). The oven temperature was programmed to increase from 50 °C (hold time 2 min) to 180 °C in increments of 20 °C/min and then from 180 °C to 330 °C in increments of 3 °C/min (hold time 10 min). The relative amount (mean value ± SD) of each component was calculated by dividing the corresponding peak area by the total HC peak area. Quantitative amounts were estimated by coinjection of nC24 as an internal standard (28 ng per sample). GC-MS analyses were performed using the GC equipment and column described for GC-FID analysis, coupled to a mass-selective detector (MSD) (Agilent model 5975C VL). The injector port was operated in splitless mode at 360 °C, and the oven temperature was programmed to increase from 50 °C (hold time 1 min) to 200 °C in increments of 50 °C/min and then to 360 °C in increments of 7 °C/min, with a holding time of 20 min. The carrier gas was helium, at 11.36 psi, with a linear velocity of 39 cm/s. The MSD was operated in the SCAN mode with a mass range of 35–850 atomic mass units, electron impact mode at 70 eV, and transfer line at 360 °C; the ionization chamber was operated at 280 °C (43). Methyl branching assignment was based on their mass fragmentation pattern and Kovats index values, estimated after injection of n-alkane standards, of 22–44 carbons (Sigma Aldrich) (43). Shorthand nomenclature to identify the HCs used in the text and tables is as follows: CXX indicates the total number of carbons in the straight chain; linear alkanes are denoted as n-CXX; the location of methyl branches is described as x-Me for monomethyl alkanes and as x,x-DiMe for dimethyl alkanes. Alkenes are shown as x-CXX:1.
Raising of Antibodies.
Rabbit polyclonal antibodies targeting a recombinant CYP4G16 peptide were made and affinity purified by Davids Biotechnologie. The sequence encoding the peptide (LRHRKMWLYPDLFFNLTQYAKKQVKLLNTIHSLTKKVIRNKKAAFDTGTRGSLATTSINTVNIEKSKSDSTKTNTVEGLSFG) was cloned using the primers Fab16 and Rab16 (Table S1), expressed in bacteria with a 6xHis tag at the N terminus, and affinity purified to homogeneity by Ni-NTA2+ chromatography. The purified peptide was used as an antigen to develop antibodies in rabbits. The specific antibodies that were used for the detection of the CYP4G17 (AGAP000877) and the CPR proteins (AGAP000500) have been previously described in refs. 18 and 24, respectively.
Immunofluorescence and Confocal Microscopy.
Three- to five-day-old sugar-fed female mosquitoes were fixed in a cold solution of 4% formaldehyde (methanol free; Thermo Scientific) in PBS for 4 h, cryoprotected in 30% (wt/vol) sucrose/PBS at 4 °C for 12 h, immobilized in Optimal Cutting Temperature (OCT) compound (Tissue-Tek; Sakura), and stored at −80 °C until use. Immunofluorescence analysis, followed by confocal microscopy, was performed on longitudinal sections of the frozen prefixed mosquito specimens as described in ref. 18.
Functional Expression of 4G P450 Cytochromes.
The 4G16-CPR and 4G17-CPR fusion protein consists of the full-length corresponding P450 cytochromes fused via a Ser–Ser dipeptide to the catalytic domain of housefly CPR (amino acids 51–671; GenBank accession no. AAA29295.1). The clone was constructed by Gibson assembly using the In-Fusion kit (Clontech). The pENTR_4G2-CPR plasmid was divergently amplified as previously described (21). The ORFs of CYP4G16PA (vector base: AGAP001076-PA) and CYP4G17 (vector base: AGAP000877-PA) codon-optimized for Sf9 cells and with flanking pENTR4R5ic and CPRfusF2ic linkers were synthesized by Biomatik. The CYP4G16PA_Sf9 and CYP4G17_Sf9 bias fragments were amplified using pENTR4R5ic and CPRfusF2ic primers (Table S1) with CloneAmp HiFi PCR Premix as above. The profile was 34 cycles at 98 °C for 10 s, 55 °C for 15 s, and 72 °C for 10 s. The pENTR_4G16PA_Sf9, pENTR_4G17_Sf9 bias fragments and the pENTR4_CPR fragment were verified by gel electrophoresis, copurified with a NucleoSpin Gel and PCR Clean-up (Machery-Nagel), assembled by In-Fusion cloning in 10-μL reactions, transformed into Stellar-competent Escherichia coli (Clontech), and selected on LB kanamycin plates. Recombinant clones were identified by PCR and sequenced to confirm the integrity of the insert. Recombinant proteins were produced in Sf9 cells using the Baculo-Direct expression system (Invitrogen), as previously described (21). CYP4G16PA-CPR and CYP4G17-CPR were quantitated by CO-difference spectrum analysis using a microplate format (21). Octadecanal (substrate) was prepared, and oxidative decarbonylase assays were performed as described in ref. 21.
Statistical Analysis.
Statistics were obtained with GraphPad Prism software, version 5.03. Differences in the total HC amounts and the thickness were analyzed with Student's t test.
SI Materials and Methods
Sequence Analysis of An. gambiae 4G Cytochrome P450.
The National Institute of Allergy and Infectious Diseases Bioinformatics Resource Center VectorBase (https://www.vectorbase.org/) was used for the initial retrieval of CYP4g sequences. The protein sequences were aligned using the CLUSTAL W alignment tool.
Extraction of RNA and cDNA Synthesis.
Total RNA was extracted from individuals or pools of female mosquitoes using TRIzol reagent (Invitrogen). RNAs were treated with Turbo DNase (Ambion) to remove genomic DNA contamination and were subsequently used to make first-strand cDNA using oligo-dT primers with SuperScript III reverse transcriptase (Invitrogen), according to the manufacturer's protocol. The primer pairs used for the amplification of the different CYP4g transcripts and the RpS7 are listed in Table S1. PCR reactions were carried out for 35 cycles using 0.15-mM primers under cycling conditions of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 30 s. RT-PCR experiments were performed using three biological and two technical replicates for each gene.
The intrathoracic injection-based methodology that previously established the efficiency of gene silencing in oenocytes of adult mosquitoes was used (24) with dsRNA derived from a cloned CYP4G16 fragment amplified by the primers Fri16 (ri) and Rri16 (ri) (Table S1). Four days later, subgroups of 10 mosquitoes for each dsRNA (dsCYP4G16 and dsGFP) were immunohistochemically analyzed. Three replicate biological experiments were performed.
Immunofluorescence on Abdominal Walls and Confocal Microscopy.
For whole-mount abdomen immunostaining, dissected abdominal walls from female mosquitoes were fixed for 30 min at room temperature in 4% methanol-free formaldehyde (Thermo Scientific) in PBS supplemented with 2 mM MgSO4 and 1 mM EGTA, washed for 5 min with PBS, and then washed in methanol wash for precisely 2 min. After the methanol wash, the tissues were washed again with PBS and then were blocked for 2 h in blocking solution (1% BSA, 0.1% Triton X-100 in PBS). Then the tissues were stained with rabbit primary antibodies in 1/500 dilution in the blocking serum (in the Triton X-100 experiment, Triton X-100 was excluded from the blocking serum), followed by goat anti-rabbit antibody (Alexa-Fluor 488; Molecular Probes; 1:1,000) that gave the green color. Finally, DNA was stained red with ToPRO 3-Iodide (Molecular Probes). In double immunofluorescence experiments, the tissues first were incubated with α-CPR (dilution 1:500), followed by goat anti-rabbit antibody (Alexa-Fluor 488) as described above, fixed again with 4% formaldehyde for 15 min, and then incubated for 1 h with labeled α-CYP4G16. A total of 3 μg of α-CYP4G16 was labeled with the Zenon kit (Alexa-Fluor 555; Invitrogen), according to the manufacturer's protocol. Pictures were obtained on Leica TCS-NT laser-scanning microscope.
SI Results
According to the VectorBase annotation, cyp4g16 has four predicted transcripts: the A, B, and C transcripts, which differ in their transcription start site but encode the same protein (CYP4G16PA), and the D transcript, CYP4G16PD, which has a predicted alternative splicing in the 3′ coding region that results in a truncated C terminus (Fig. S1A). Analysis of RT-PCR products confirmed the predictions for splicing of the fourth intron of the A-, B-, and C-type transcript. However, the presence of the cyp4g16PD-type spliced form was not confirmed; a transcript was found that was unspliced in this region, and thus contained the predicted fourth intron of the gene, resulting in an alternative reading frame that encoded a longer protein form, which we renamed "CYP4G16PD1." However, transcription of cyp4g16RD1 appeared very low in comparison with cyp4g16RABC when examined by RT-PCR (Fig. S1B).
Furthermore, the intriguing association of CYP4G16 protein with the PM needed further investigation. We found that the signal was specific for α-CYP4G16, because knock down of the corresponding gene by RNAi greatly diminished fluorescence in the oenocytes (Fig. S2A). Furthermore, differential permeabilization (Fig. S2B) indicates that Triton X-100 treatment of sections is necessary for efficient staining (25), suggesting that the recognized CYP4G16 epitopes are inside the cell, and thus the PM-associated protein faces the cytoplasm. Finally, a double-staining experiment (Fig. S2C) showed a partially overlapping signal (yellow) between α-CYP4G16 (red) and α-CPR (green), indicating a possible physical association between the corresponding proteins.
We thank Dr. Evangelia Morou (University of Crete) and Fotini Tanka for help with initial experiments on the uptake of radiolabeled deltamethrin and the stainings, respectively; Sevasti Papadogiorgaki for help with TEM; and Prof. Feyereisen (University of Ghent) for helpful suggestions during this project. This work was funded by the European Union Seventh Framework Programme FP7 (2007–2013) under Grant Agreement 265660 AvecNet.
- ↵1To whom correspondence may be addressed. Email: janet.hemingway{at}lstmed.ac.uk or vontas{at}imbb.forth.gr.
Author contributions: V.B., A.K., M.M., C.T., S.J.M., A.A., A.L., and S.A. performed research; V.B., G.J.B., M.P.J., H.R., G.J.L., and J.V. designed research; M.P.J., S.J.M., and G.C. contributed new reagents/analytic tools; and V.B., G.J.B., M.P.J., G.C., J.H., H.R., G.J.L., and J.V. wrote the paper.
Reviewers: J.B., University of Florida; B.D.B., National Institute for Communicable Diseases; and P.M., University of Basel.
The authors declare no conflict of interest.
See Commentary on page 9145.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1608295113/-/DCSupplemental.
Freely available online through the PNAS open access option.
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text:
A Nice Girl Like Me 1969
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Candy is a fetching unwed young lady with a penchant for pregnancy. Her adventures begin when she leaves her sheltered boarding school background for Paris. The result is the birth of Valentine nine months later.
Starring: Gladys Cooper Miriam Margolyes
Links: Posters, IMDb
Links: Posters, IMDb


text:
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Click on my Facebook tab above if you'd like to watch me each week on my Live Facebook broadcast each Sunday afternoon around 3 p.m. PST.Newgin Godzilla Figure B Set Novelty Make sure to "like" my Public Facebook page so you can watch it on my page or yours.
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text:
A Touch Mortal by Leah Clifford
Release Date: Already released! (February 22, 2011)
Publisher: Greenwillow Books
Format: Paperback, 419 pages
Source: Won from Justin's Book Blog!
Description from Goodreads:
Eden didn't expect Az.
Not his saunter down the beach toward her. Not his unbelievable pick up line. Not the instant, undeniable connection. And not his wings.
So long happily-ever-after.
Now trapped between life and death, cursed to spread chaos with her every touch, Eden could be the key in the eternal struggle between heaven and hell. All because she gave her heart to one of the Fallen, an angel cast out of heaven.
She may lose everything she ever had. She may be betrayed by those she loves most. But Eden will not be a pawn in anyone else's game. Her heart is her own.
And that's only the beginning of the end.
This book drew me in for a few reasons. First, the description is AMAZING. Second, I have heard many, many, many good things about this book. (The most prominent of these good things came from Kreag at I Devour Books.) And lastly, this book was more than 400 pages. What can I say? I like it when books are long enough to not end the day I start them. When adding all of this up to create my expectations for this book, I'd say that they were pretty high. I mean, I REALLY value other people's opinions when it comes to what I should and should not read. I am super happy to say that my expectations were certainly met, if not exceeded.
I thought this book would be based heavily on the whole "love" angle. Lots of books are, so really that's a realistic assumption. And I was half right. Love was somewhat important in this book, but it was not what made this story. The love had to be there, yes, but it was not really the main focus. We paid much more attention to Eden's predicaments with the Fallen and the other Siders than we did to her feelings for Az most of the time. Still, the author swooped in at just the right times to push Az back into our minds so we don't forget him.
Oh, the issues faced in this book. They thoroughly amused me and held my attention for all 400 pages. I remember how I was nearing the end of the book and hoping for more pages to magically appear so the book would not end. If that is not a sign that a book was great, I don't know what is.
Character rundown: Eden. I liked Eden! I loved how independent she was, not really reliant on her love interest like a lot of other heroines are in YA books. She does things for herself and controls what she wants to and doesn't care about anything else, and I love that. A kick-booty girl who actually takes charge. Oh, how I have waited for a girl like her in a book. Now, Az. OH, AZ. I adore Az, as I usually do with the guys in books. I love how he puts Eden first and doesn't give up on her. I tell you, it is things like these that melt my heart and win me over EVERY TIME. And it never gets old. Last, I'll say a few things about Luke. I have to admit that I swoon for Luke. The way he is described, he is a true heartthrob. I love that he is 100% evil, though. Kind of balances out his beauty. And his love. Yes, evil people find love as well. Or at least, I assume he loved her. That's what I got from certain happenings in this book. ;)
As a whole, this story was purely gorgeous. Yes, I described a STORY as gorgeous. Because this one really was. I am immensely glad I got this book, and I encourage you all to read it, if you're into the whole fallen-angels thing. . . . On second thought, even if you're not. I say anyone should give this book a try because it is not your everyday angel story. It's different, and I enjoy that! It's always nice to mix up the angel story that is so common these days.
Okay, that's it, Friends! Before I bid you all good day, though, I want to send a thousand thanks to:
Justin from Justin's Book Blog - DUDE. I love your blog! I visit, like, every day. And I tend to comment a lot. But still, your blog is on my list of favorites. Thank you so much for this book!
Kreag from I Devour Books - Because you're just awesome. Seriously, your best friend badge is ULTRA shiny right now. Yeah, even shinier than mine. That is an accomplishment, my friend. Thank you for being first to steal my blog button!
All right, now that's really it, folks. Thank y'all for reading my reivew and visiting my blog!
Stay safe until next post, Friends!


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Bespoke Wedding Rings
Wedding rings can be custom made to complement all engagement rings.
Commission a bespoke wedding ring direct from fine jewellery manufacturers in London's Hatton Garden.
Wedding rings can be set with diamonds or precious gemstones.
Men's wedding rings are also available.
Contact us to discuss your designs, or make an appointment to view and try on examples of both traditional and unusual wedding rings.
Your wedding ring can be shaped to gently curve around your engagement ring.
Choose platinum wedding rings, 18 carat yellow gold or 18 carat white gold wedding rings. The style can be either court, D-shape or flat.
Home | Who We Are | Custom Jewellery Design Service | Visit Us | Contact Us
How to Order | How to Pay | Delivery Times | Shipping | VAT and Taxes | Return Policy
|Copyright � 2003-2015 Engagement Rings of London UK
|We are open Monday to Friday from 09.30 to 17.30. By appointment only.
|100 Hatton Garden at The London Diamond Bourse, London, EC1N 8NX, UK
|Telephone 020 7193 4412 or e-mail to make an appointment.


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In a career that has been punctuated by uncomfortable, emotionally freighted spectacles, Hillary Rodham Clinton's news conference on Tuesday explaining her use of private email to do her job as secretary of state was far from the most gawk-worthy.
She began by opining on the diplomatic furor of the day, a reminder that in the four years she was typing out missives on her smartphone, she was also representing America's national interests around the globe. Her voice measured, she conceded at the outset that it probably would have been "smarter" not to work from a private email account. And she speculated that ordinary Americans would understand her concern for keeping private matters private, like her yoga poses or her mother's funeral.
But Mrs. Clinton's attempt to put a whirlwind of questions and critical news reports behind her — and to get back to mustering enthusiasm ahead of her expected announcement that she will make another run at the White House — devolved, over the course of 21 minutes, into an exchange of sharp-toned questions and increasingly defensive responses, both in what she said and in her demeanor in saying it.
Standing at the United Nations with a tapestry of Picasso's "Guernica" behind her, Mrs. Clinton frequently lowered her eyes to the notes she had positioned before her in a black binder, or looked off into the middle distance. She resorted to lawyerly argument, insisting that she had followed all the relevant laws, rules and regulations. She offered new assertions and details for Republicans and journalists to chew over.
Above all, she asked the public to trust her: to take her word that the 30,000-odd emails that she said she had deleted were truly personal, not professional or political; truly private, not merely embarrassing.
"No one wants their personal emails made public, and I think most people understand that and respect that privacy," she said.
Mrs. Clinton, who has seemed to have a phobia about being treated like a candidate too soon, avoided directly answering questions about how the email issue had affected her thinking about 2016.
And yet over the course of her brief appearance, which several cable television networks carried live, she seemed to become a presidential candidate again in real time: She was fielding questions in a defensive posture for the first time in two years.
She was doing, in other words, what candidates under fire must do. And she did not look happy.
Mrs. Clinton has done well under other high-profile, stressful moments. In 1994, she held a 66-minute news conference to deal with mounting questions about the Whitewater land deal.
"I do feel like I've always been a fairly private person leading a public life," she said at the time, explaining that this had led her "to perhaps be less understanding than I needed to of both the press and the public's interest, as well as right to know, things about my husband and me."
In the view of Mrs. Clinton and her allies, the news media and the public will be getting even more than they are entitled to from her email correspondence at the State Department. The emails she has turned over to be vetted and then publicly released went "above and beyond" what was required of her, she said, and would be enough to provide what she called "unprecedented insight" into the work and life of a cabinet member.
Her implication was not an unheard-of argument from her: that she was being held to an unfair standard, beyond what is expected of other federal officials.
Mrs. Clinton worked hard to set the terms of Tuesday's news conference. It was held at the United Nations after she had given a much-ballyhooed speech at a conference on women's rights. She quickly swiveled to what she called "a matter in the news today," but then made it clear that she was speaking of the latest effort by Senate Republicans to thwart a nuclear deal the Obama administration is negotiating with Iran. She aligned herself firmly with Mr. Obama, briefly putting herself back in familiar partisan territory.
But while her delivery and tone were steady, the new information Mrs. Clinton revealed — that she had deleted around half of her emails from her time as secretary of state — made it impossible for her to put the subject to rest for good.
"Looking back, it would've been better if I'd simply used a second email account and carried a second phone, but at the time, this didn't seem like an issue," Mrs. Clinton said, a refrain she returned to throughout the news conference.
Within Mrs. Clinton's circle, several people had urged her to respond faster to the email story, perhaps on television with a friendly interviewer. Others expressed hope that she would decide that it was time to build the kind of aggressive media operation that an out-and-out campaign demands.
In any case, this was not to be the sort of news conference that Gov. Chris Christie of New Jersey gave after reports that an aide had been involved in lane closings at the George Washington Bridge.
It had taken eight days for Mrs. Clinton to make herself available for questions. And long before the questions ran out, she began packing up her binder.
As a reporter asked about a former ambassador to Kenya who was fired over infractions that included using personal email for business, Mrs. Clinton signaled that she was done.
"That is not an accurate representation of what happened," she said to the reporter, David Shuster, a journalist who made a derogatory statement involving Mrs. Clinton's daughter, Chelsea, during the 2008 presidential campaign.
As others called out more questions, she said, "Thank you!" and walked off.


text: Curtis P. Bounds has been appointed by the Delaware State Bar Association to serve on its Fee Dispute Conciliation and Mediation Committee. In this position, Curtis will participate in resolving fee controversies between lawyers and clients and between lawyers who have represented the same client. Proceedings before the Committee may be initiated by or on behalf of a lawyer or client. The Committee conducts an informal hearing and at its conclusion of the hearing, the Committee shall issues an opinion related to the dispute.


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Don't mind the mess!
We're currently in the process of migrating the Panda3D Manual to a new service. This is a temporary layout in the meantime.
A DirectScrollBar is similar to the "scroll bar" widget commonly used by the user to page through a large document. DirectScrollBar is available beginning in Panda3D 1.1. It consists of a long trough, a thumb that slides along the trough, and a pair of buttons on either side of the trough to scroll one line at a time. A DirectScrollBar can be oriented either vertically or horizontally.
The DirectScrollBar is similar in function to DirectSlider, but it is specifically designed for scrolling through a large window. In fact, a pair of DirectScrollBars is used to implement the DirectScrolledFrame, which manages this scrolling functionality automatically. (Because DirectScrolledFrame exists, you will probably not need to create a DirectScrollBar directly, unless you have some custom purpose that requires a scroll bar.)
DirectScrollBar has many things in common with DirectSlider. Like DirectSlider, the normal DirectGui parameters such as frameSize, geom, and relief control the look of the trough. You can control the look of the thumb by prefixing each of these parameters with the prefix "thumb_", e.g.
thumb_frameSize; similarly, you can control the look of the two scroll buttons by prefixing these with "incButton_" and "decButton_". You can retrieve or set the current position of the thumb with
|Initial position of the thumb
|Default is 0
|The (min, max) range of the thumb
|Default is (0, 1)
|The amount to jump the thumb when the user clicks left or right, (up or down if the scrollbar is vertical), of the thumb; this also controls the width of the thumb when resizeThumb is True
|Default is 0.1
|The amount to move the thumb when the user clicks once on either scroll button
|Default is 0.01
|The orientation of the scroll bar
|DGG.HORIZONTAL or DGG.VERTICAL
|Whether to automatically adjust the buttons when the scroll bar's frame is changed
|True or False
|Whether to adjust the width of the thumb to reflect the ratio of pageSize to the overall range; requires manageButtons to be True as well
|True or False
|Function called when the position of the thumb changes (takes no arguments)
|Extra arguments to the function specified in command
|[Extra Arguments]
|thumb_geom, thumb_relief, thumb_text, thumb_frameSize, etc.
|Parameters to control the look of the thumb
|Any parameters appropriate to DirectButton
|incButton_geom, incButton_relief, incButton_text, incButton_frameSize, etc.
|Parameters to control the look of the lower or right scroll button
|Any parameters appropriate to DirectButton
|decButton_geom, decButton_relief, decButton_text, decButton_frameSize, etc.
|Parameters to control the look of the upper or left scroll button
|Any parameters appropriate to DirectButton
Here is a small example on how to create the scrollbar:
from direct.gui.DirectGui import DirectScrollBar mybar = DirectScrollBar(range=(0, 100), value=50, pageSize=3, orientation= DGG.VERTICAL) mybar.setPos(-1, 0, -0.5)
This will give you a scrollbar at the lower left side of the screen. If you want to parent the scrollbar to a determined frame, you add the keyword parent to the set of keyboards like so:
mybar = DirectScrollBar(parent=myframe, range=(0,100), value=50, pageSize=3, orientation= DGG.VERTICAL) mybar.setPos(-1, 0, -0.5)
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Apples and Oranges – Rocks and Pears, Schools and Homeschools
Have you ever thought about what makes homeschooling such a unique approach to living and learning with our children? Have you ever considered why it changes our lives in ways no other educational option can? Helen Hegener, HEM M/J 2004.
Debunking the myths of home schooling
With one to two million American children learning outside of school, home schooling can no longer be considered an extremist position or fad. Home schooling is a valid and effective educational option. By Lisa Rivero.
The Homeschool Alternative
Home based education legal in all 50 states — is a growing movement that offers parents and children alike the opportunity to grow and learn in a loving and supportive environment. By Kris Bordessa.
Homeschooling, by Patricia M. Lines
Choosing to homeschool or to traditionally educate a child is often a difficult and confusing decision for parents and guardians. To help them make the best choice possible, this brochure answers basic questions about homeschooling and suggests other useful sources of information.
Homeschooling Works
There are a number of reasons why homeschooling works, and why we must protect the diversity of homeschooling practices to continue this success.
I Was Homeschooled and I Just Didn't Know It
After recently hearing about home schooling and reading some of the articles and philosophy of this wonderful way of life, I realized that, in fact, I had been home schooled, and that is the reason I am a well educated person today. by Elaine Edwards.
The Moore Formula
Properly done, home education offers low-stress, low-cost, and high achievement and sociability from a balance of study, work, and service. Few curricula do this! Dr. Raymond Moore, founder of the modern homeschool movement.
Parents Deciding and Directing
Homeschooling is parents deciding and directing the education of their children. Deciding and directing the education, not doing all the educating. From At Home in America.


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GAF® has a pocketful of intuitive programs to make home designing a picnic. One of its more popular tools is the Virtual Home Remodeler, a handy companion to start your exterior remodeling project the easy way.
As one of the few GAF Master Elite™ roofing companies in Northern Virginia, Peak Roofing Contractors is thrilled to share with you the wonders of the Virtual Home Remodeler and help you unleash the exterior home designer in you.
Here's how it works:
Leaving Little to the Imagination
Unlike browsing through an extensive selection of GAF shingles and imagining how each would look like on your home, this tool would give you a lifelike impression of your new roof in just a few clicks. Complete with GAF's collections of products, you can easily get a glimpse the distinct features of every shingle in a variety of colors.
This program is very similar to Roofing Wizard, only it allows you to see how your replacement roofing in Manassas would blend with your trim and brick or siding. Whether you're using a sample or an actual image of your home, you can change the colors and textures with countless options.
Make One or One Dozen Creations
The Virtual Home Remodeler lets you save several projects. This means you're not limited to creating just one design. You're free to make one masterpiece after another for as long as you want, allowing you to compare and contrast your projects later on.
Hear What the World Thinks
This tool is highly interactive. You can quickly share your creations through email, Facebook, and Twitter. This way, your friends can validate or help you improve your work. You can also print and show all your designs to us so that we can see how exactly you envision your Manassas roofing system.
Try the GAF Virtual Home Remodeler now! If you're ready to share your creations with Peak Roofing Contractors, just call (703) 745-5153 at your most convenient time.


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All Rainouts, Cancellations: Manager will need to contact Tom Fitzgerald or send an email to email@example.com.
Games are subject to change without notice. The District 1 website for Interleague Game Schedule is the Official Schedule. Please check it often - www.littleleaguewad1.org
It is the home team's responsibility to contact District to reschedule the games in Stack Sports.
Click below for District 1's Interleague Schedules
BB Juniors and Tball schedules coming soon.


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You have to wonder just how far from its best self the United States has wandered when one of the nation's leading war criminals is nervous about the incoming administration. From Foreign Policy:
"He thinks all kinds of crazy things about prosecutions," said John Yoo, a Berkeley law professor who, while serving at DOJ's Office of Legal Counsel in 2002 and 2003, helped write legal justifications for aggressive interrogation methods that critics call torture. Those memos have since been rescinded. "I don't think he has a very good sense of how our law enforcement system works," Yoo told Foreign Policy.
The proponent for theoretical prepubescent ball crushing thinks El Caudillo del Mar-A-Lago is an ignoramus and a threat to the rule of law. Let that roll around in your head for a while. Get up from the floor only when you feel it's safe for you to stand. For myself, every day that I wake up and discover that the name of some notorious atrocity serial killer hasn't been floated for Surgeon General, I feel temporarily relieved. Then, I open the other eye.


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A claim like "world's best protection"
Dubbing 3rd Rock the Holy Grail of UVA protection was quite an endorsement, especially coming from an industry specialist of Joe Stanfield's standing. After 40 years in the business, Joe made the comment once he'd seen the results of in vitro tests comparing 3rd Rock's absorbance to those of two leading sunscreens (below).
As you can see, all three products start out high on the UV light absorbance axis at 290 nm, the low end of the UVB spectrum (290–320 nm). But while the other two brands begin breaking down almost immediately (click here for why), 3rd Rock absorbance continues unchanged across the UVB spectrum and well into the UVA range (320–400 nm). Then, instead of decreasing, from about 350 to 375 nm, absorbance actually increases.
Nonetheless, the crucial point is the consistency of that performance. 3rd Rock's UV light absorbance—a.k.a. broad-spectrum UVA/UVB protection—shows no decrease over the 290-375 nm wavelength range. And that's something no other sunscreen has ever come close to.
3rd Rock's "impossible" UVA/UVB ratio and 5-star
Assumptions about ratio limits went out the window as 3rd Rock's 350-375 nm "performance surge" sent our UVA/UVB ratio soaring past 1, culminating in Joe Stanfield's Holy Grail pronouncement. After all, no other sunscreen we know of can claim a ratio higher than .75.
Boots awards three stars (★★★) to sunscreens with UVA/UVB ratios from 0.61 to 0.8 and four stars (★★★★) to those between 0.81 and 0.9. Five stars (★★★★★), the highest rating, requires a UVA/UVB ratio greater than 0.91 and indicates performance closest to uniform UV protection.
3rd Rock offers the only 5-star UVA protection on today's market.
©2010 3rd Rock Sunblock | Website designed by Nick Romanos


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The sports hall is ideal for a range of sporting and non-sporting events and functions.
Trampolining courses are available for children from the age of 5 years and above. Each course is available to children of various abilities - whether a complete beginner or more advanced. Plus, every child gets their first session as a free trial!
All *Squad* groups are by invite only.
For more information please contact Kestrel 07949 223368
Or email: firstname.lastname@example.com
Website: www.kestrelgymnasticsacademy.co.uk


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I would also like to add to this that whoever coordinates the music should not only avoid depressing music but also uber inappropriate rap. Since I came only as a coach this year, I sat with several of the parents from my team. Several were disgusted by the lyrics of some of the songs, which included all kinds of things they had not discussed with their sixth graders. I'm not saying to play nursery rhymes, but next time there needs to be some consideration that there are still children under the age of 13 at nationals and any music played should respect that.And the Parade of States was a little sad for the teams at the beginning. Marching out to empty seats (and the sad music...) was kind of depressing. Also, it took forever for everyone to get seated and there was this massive bottleneck in the middle as a result. Because of that, there were also long pauses between each school getting called out, and a lot of the crowd's energy was lost when the guest speakers finally came. Dr. Gay was cool; the music and the videos were really awkward. All in all, next time, please, don't have all 15 people marching. Plus, if a state has two teams each, don't try to cram all four of them into one room.
tiny.cc/sontWHos got the link to scoresheet for division C?
The scoresheets are also available on the 2015 National Tournament page at soinc.org.tiny.cc/sontWHos got the link to scoresheet for division C?
Its a combined score sheet, scroll down for division C
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text: Share: Comments Print June 26, 2012 at 10:14 PM Update: Sounders will host Chivas USA in a U.S. Open Cup semifinal on July 11 Posted by Joshua Mayers Update 12:05 a.m. Friday — Information in this posted has been updated to reflect a date change. Sounders FC has advanced to the semifinals of the U.S. Open Cup after a hard-fought 1-0 road win Tuesday against the San Jose Earthquakes. Here are the details on Seattle's next step toward a historic four-peat, which would be the first in the tournament's 99-year history. Chivas USA (MLS) at Seattle Sounders FC (MLS): Starfire Stadium; Tukwila, Wash.; 7 p.m. PT, July 11 Tickets go on sale at 10 a.m. Friday. This is the sixth-straight year Seattle will have representation in the Open Cup semifinals. Sounders FC has qualified the last four years (2009-10) and the Sounders of the United Soccer Leagues also made it to the final four a couple times (2007-08). * * * Any thoughts on the matchup? Comments


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Search Results
This floor needed resilience, so we brought it. Polishing 100+ year old concrete can be challenging. We brought our specialty tools and techniques to ensure this concrete floor stays polished for another 100+
Calvin Klein needed a simple yet elegant look for their floor, so we did just that.


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International Panorama 2
●2nd November 18:15 Theater3
Director : Vladislav Knezevic
2014 / Croatia / 0:12:45 / Bonobostudio / Bonobostudio
The fall of a device named A.D.A.M. (Autonomous Drone for Asteroid Mining) causes the strengthening of its cognitive processes and activation of the autonomous mode. This leads to its refusal of communication with the satellite company operators.
Vladislav Knezevic
Vladislav Knezevic (1967) graduated from the Academy of Drama Arts (TV and Film Direction Dept.) in Zagreb and De Vrije Academie (Audio-visual Dept.) in Den Haag. Professionally works as a freelance director for TV and various productions since 1993. He is into experimental film, video and sound processing since 1988. He has also initiated and organized several film programmes and presentations (Reference to Difference, Videodrome TV edition, 25 FPS Festival).
Tears of Inge
Director : Alisi Telengut
2013 / Canada / 0:04:21
A profound human-animal and human-nature relationship is represented by a painted world filled with a camel's emotion and tears. It is based on a real Mongolian nomadic story narrated by my grandmother.
Alisi Telengut
Alisi Telengut, born in 1989, creates animation frame by frame under the camera, with painting as the medium, to generate movement and explore hand-made and painterly visuals for her films. Her works have been culturally engaged and focus on nomadic ethnic minorities. Her recent films received awards, have been screened in various worldwide film venues and exhibitions as moving image art works, and have also contributed to enthnographic, ethnocultural and archaeological research.
Yùl and the Snake
Director : Gabriel Harel
2015 / France / 0:13:11 / Kazak Productions / L'Agence du court-métrage
Yùl, 13 years old, goes with his big brother Dino to conclude a deal with Mike, a thug escorted by his Argentinian mastiff. When things go wrong, a mysterious snake appears.
Gabriel Harel
Gabriel Harel has studied at the Epinal School of Fine Arts for four years. In 2007, he enters the school La Poudrière in which he directed "L'Eau à la bouche", a traditional animation film. "Yùl and the Snake" is his first short film, produced by Kazak Productions.
The Meek
Director : Joseph Brumm
2015 / Australia / 0:07:26 / Studiojoho
The Meek, an original animated short created by StudioJoho's Joe Brumm, Mark Paterson and Laura DiMaio, follows the story of a very small person trying to quit a very big bad habit. When her cold, lonely existence is changed forever by the arrival of a huge comet and a smouldering stranger, our heroine thinks her luck has changed. But when things sour she quickly finds out that knowing what's good for you doesn't always mean doing what's good for you. Made using digital cut-out,with Myf Warhurst narrating and featuring a beautiful score by The Cat Empire's Ollie Mcgill, The Meek is both tiny and grand, toxic and detoxifying.
Joseph Brumm
Joe Brumm worked as an animator for 10 years in London on numerous children's series, including the Bafta winning Charlie and Lola. After returning to Australia he founded Studiojoho which now has an expanding body of quality work behind it. It's clients include CollegeHumor, ABC's Good Game, GOMA and Halfbrick. The Meek is Studiojoho's first funded short film.
Director : Nils Hedinger
2014 / Switzerland / 0:05:35 / prêt-à-tourner / KFA Hamburg
A group of logs is about to freeze to death in a cold, icy desert. When they realize that the only fuel for a warming fire is their own body, things start heating up.
Nils Hedinger
Nils Hedinger was born in Berne, Switzerland in 1986. He studied animation in Lucerne and makes his own films since graduating in 2010.
Director : Antje Heyn
2015 / Germany / 0:07:39 / Protoplanet Studio / Protoplanet Studio
PAWO (Tibetan for "being brave") is the magical adventure of a little toy figure, who finds herself in a curious world. Thanks to some strange companions, she gradually becomes aware of her strength and skills.
Antje Heyn
Antje Heyn (1979 in Bergen auf Rügen) is a Berlin based animation director and illustrator. Her work is inspired by daily observations of urban living and culture as well as nature and animals. Besides setting her heart on creating characters and bringing them to life she teaches animation at art academys and is running the Protoplanet Studio.
Director : Dana Sink
2015 / United States / 0:01:16
I want to ride my bicycle.
Dana Sink
Dana Sink lives in Harrisburg, Pennsylvania with his wife, daughter, three cats, one dog, and a frog. Recently, Dana has drawn inspiration from his three year old daughter and creating allegorical animations that she can relate to has become a priority. His paintings, film, and animation have been seen in galleries and museums across America. His achievements include several group and independent exhibitions, being featured in various articles and publications including a book on Savannah, and receiving awards for his paintings in various exhibitions. His films have received recognition in several film festivals and his work has been seen on MTV and MSNBC.
Director : Tess Martin
2014 / Netherlands / 0:02:53
In Italian playgrounds a song is chanted that dates back to World War I. This paint on glass animation tells this dark tale of a soldier who returns home from war to find his girlfriend has left him.
Tess Martin
Tess Martin is an independent animator who works with cut-outs, ink, paint, sand or objects. Her most recent award-winning films are The Lost Mariner, an animated interpretation of an Oliver Sacks case study, and Mario, a paint on glass film based on an Italian folk song. Her films have displayed at galleries and festivals worldwide. She curates touring programs of films from Seattle and the Northwest and runs a monthly animation event in Rotterdam, the Netherlands. She also occasionally writes about the world of independent animation.
Director : Josh Shaffner
2015 / United States / 0:02:55 / CalArts
'Flaws' is a hand drawn animated short about a service industry worker and the bitterness that stews in the under stimulated and over worked. The film utilizes a unique animation technique in which all frames from the animated sequences are drawn on a single sheet of paper. It is a companion piece to 'Service' which is also being submitted to the festival.
Josh Shaffner
Josh Shaffner is a film maker and visual artist. He received his BFA in Painting and Drawing from The University of Montana 2005 and his MFA in Experimental Animation from CalArts in 2015. His work has screened in festivals world wide and shown in museums and galleries throughout the United States and Australia. He currently resides in Los Angeles, California where he works as an independent artist and animator.
Hallgrimur og Jeremy
Director : Jeanette Bonds
2014 / United States / 0:02:30 / GLAS Animation
Two boys discover violence. One discovers regret.
Jeanette Bonds
Jeanette Bonds is a writer and independent animator living in Los Angeles. She is co-founder and director of GLAS Animation, an animation festival and resource for, innovative, and independent animators across the globe. Her films have been featured at Encounters Short Film and Animation Festival, London, Seoul, Holland, and Melbourne International Animation Festival, and in 2014 was nominated for an Annie Award.
Director : Marc Riba, Anna Solanas
2013 / Spain / 0:17:00 / I+G Stop Motion
Teo survives isolated in a house constantly besieged by a horde of stray dogs.
Marc Riba, Anna Solanas
Marc Riba and Anna Solanas have been working in the world of puppet animation for 12 years. They have made 7 short films which have been selected in more than 1000 festivals around the world.


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4 17968 56A Avenue, Surrey Listing #: R2405224
TIRED of TINY TOWNHOMES? Well look no further. This is 1 of the biggest ones out there with over 1900 sq ft, that allows room for all sizes of furniture. Bright END UNIT with lots of windows and skylight. White kitchen w/abundant counter space & pantry Adjacent Eating Area & Dng Rm. Large Lvg Rm with Stylish Fireplace Mantel. HUGE Master Bdrm w/Vault Clng. LARGE Bdrm down with own Bath perfect for teens or in-laws. MASSIVE sundeck perfect for entertaining. Lots of storage. Plus a rarity in strata - TONS of pkng with room for 6 vehicles and additional pkng on the QUIET dead-end street. Close to shopping and TRADITIONAL SCHOOL. Open to all ages and pets with restrictions.
Taxes (2019): $2,337.07
- Clothes Washer
- Dryer
- Dishwasher
- Disposal - Waste
- Drapes
- Window Coverings
- Garage Door Opener
- Pantry
- Refrigerator
- Security System
- Stove
- Vacuum - Built In
Site Influences
- Central Location
- Cul-de-Sac
- Shopping Nearby
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Listed By: RE/MAX Little Oak Realty (Abbotsford)
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