text:
The development of new drugs and vaccines against malaria poses one of the major challenges to current medical research. It must be grounded in the thorough understanding of the parasite's biology, including its interactions with the host, and the Anopheles mosquitoes that transmit it. P. berghei provides a highly tractable model to study the fundamental cell biology of malaria parasites and many aspects of their interactions with host and vector.
We are particularly interested to learn how the cellular processes involved in sexual development and mosquito transmission are regulated through signal transduction pathways that control stage conversion, gene transcription and protein translation. We discovered that parasite development in the mosquito is triggered by xanthurenic acid (Billker et al., 1998), a tryptophan metabolite, which in insects is present at high concentrations. It regulates parasite development through a plant like protein kinase, which we identified by experimentally disrupting the parasite gene encoding it (Billker et al., 2004). Working with our collaborators we recently demonstrated that a drug-like chemical inhibitor of the kinase can block malaria transmission to mosquitoes when administered to a mouse (Ojo et al., 2012).
The targeted modification of the genome is far more difficult in malaria parasites than in model organisms, such as yeast. To increase the rate at which discoveries can be made in P. berghei, we have developed protocols to produce more efficient genetic modification vectors and other molecular tools that allow researchers to rapidly switch parasite genes on and off. We now operate a production pipeline to produce gene knock out and tagging vectors for every gene in the P. berghei genome. These are freely available to all researchers and can be viewed through the PlasmoGEM database. Our vision is that over the coming years, protocols will be developed to use PlasmoGEM vectors in genetic screens that query the functions of hundreds or thousands of parasite genes at the same time. PlasmoGEM is a joint project with the Rayner group. It relies heavily on the reference genomes produced by Matt Berriman's Parasite Genomics team.
Our aims
Tools and resources for P. berghei genetic modification.
A small team, now led by Ellen Bushell, runs the PlasmoGEM project. We have generated arrayed libraries of P. berghei genomic DNA with average inserts of ca. 8.5 kb. These are large inserts, considering that P. berghei DNA is very AT-rich, making it highly unstable in E. coli. We also developed a set of tools and protocols to engineer genomic DNA in E. coli with the help of transiently expressed phage recombinases, a process termed recombineering. These protocols are robust enough to work on 96-well plates. They allow us to turn library clones into complex genetic modification vectors at scale. Using this technology we are now producing genome-wide sets of genetic modification vectors and make these available as a free resource.
PlasmoGEM vectors have advantages over conventional designs: due to their long homology arms they integrate very efficiently. Furthermore, since the vectors never exist in a circular form, targeting essential genes does not select for episomes, thereby eliminating a major source of false positive results. We are exploring the new opportunities offered by these advantages. We have also developed tools for the insertion of site-specific recombination sites for Flp recombinase, and with the Soldati and Llinas labs we produced a tetracycline repressible transactivation system to knock down essential genes in P. berghei blood stages.
We are keen to transfer the new genetic technologies to the human malaria parasite, P. falciparum. However, this is not straight forward, since our recombineering pipelines relies on keeping DNA linear for increased stability in E. coli. In contrast to P. berghei, transfection of P. falciparum relies on circular DNA.
Signal Transduction Pathways Regulating Sexual Development
Sexual development in Plasmodium is tightly linked to transmission by mosquitoes. In P. berghei sexual stages can be cultured in vitro and are relatively tractable for genetic and biochemical studies, allowing us, for instance, to identify Hap2 as a gamete membrane protein which probably has a conserved function in gamete fusion diverse eukaryotes. We also investigate the roles of protein kinases and second messengers in regulating sexual differentiation. A recent study on a plant like calcium dependent kinase, CDPK1 (Sebastian et al., 2012) illustrates how we have begun to use molecular phenotyping of to understand gene function. Various project now generate a deep understanding of some of our mutants through comparative analysis of transcriptomes, proteomes and secondary protein modifications, such as phosphorylation.
Host Genes Regulating Parasite Development
There is an urgent need for new experimental models that can identify the role of host genes in parasite-host interactions. As genome-wide association studies discover more of the relevant natural genetic variation in human populations that controls susceptibility to malaria, new tools to identify the underlying molecular mechanisms will be required. Some projects in the lab explore the use of knock out mice, in vitro differentiated mouse embryonic stem cells and human iPS cells to get at this problem.
The PlasmoGEM website hosts a database of P. berghei large insert genomic clones, genetic modification vectors and recombineering protocols.
To learn more about Malaria Experimental Genetics, consider applying for the Wellcome Trust Advanced Course we run with Julian Rayner's team each year.
Selected Publications
Recombination-mediated genetic engineering of Plasmodium berghei DNA.
Methods in molecular biology (Clifton, N.J.) 2013;923;127-38
A Plasmodium calcium-dependent protein kinase controls zygote development and transmission by translationally activating repressed mRNAs.
Cell host & microbe 2012;12;1;9-19
PUBMED: 22817984; PMC: 3414820; DOI: 10.1016/j.chom.2012.05.014
Transmission of malaria to mosquitoes blocked by bumped kinase inhibitors.
The Journal of clinical investigation 2012;122;6;2301-5
A scalable pipeline for highly effective genetic modification of a malaria parasite.
Nature methods 2011;8;12;1078-82
PUBMED: 22020067; PMC: 3431185; DOI: 10.1038/nmeth.1742
Cutting edge: the membrane attack complex of complement is required for the development of murine experimental cerebral malaria.
Journal of immunology (Baltimore, Md. : 1950) 2011;186;12;6657-60
PUBMED: 21572031; PMC: 3110530; DOI: 10.4049/jimmunol.1100603
The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission.
Cell host & microbe 2010;8;4;377-87
PUBMED: 20951971; PMC: 2977076; DOI: 10.1016/j.chom.2010.09.006
A cyclic GMP signalling module that regulates gliding motility in a malaria parasite.
PLoS pathogens 2009;5;9;e1000599
PUBMED: 19779564; PMC: 2742896; DOI: 10.1371/journal.ppat.1000599
Calcium-dependent signaling and kinases in apicomplexan parasites.
Cell host & microbe 2009;5;6;612-22
PUBMED: 19527888; PMC: 2718762; DOI: 10.1016/j.chom.2009.05.017
The conserved plant sterility gene HAP2 functions after attachment of fusogenic membranes in Chlamydomonas and Plasmodium gametes.
Genes & development 2008;22;8;1051-68
PUBMED: 18367645; PMC: 2335326; DOI: 10.1101/gad.1656508
Calcium and a calcium-dependent protein kinase regulate gamete formation and mosquito transmission in a malaria parasite.
Cell 2004;117;4;503-14
Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito.
Nature 1998;392;6673;289-92
Team members
- Mathieu Brochet
- unknown
- Burcu Bronner Anar
- Advanced Research Assistant
- Ellen Bushell
- firstname.lastname@example.com Staff Scientist
- Gareth Girling
- Advanced Research Assistant
- Stefano Iantorno
- PhD Student
- Kasia Modrzynska
- email@example.com Scientist
- Ruddy Montandon
- Postdoctoral Fellow
- Frank Schwach
- email@example.com Computational Biologist
- Jaishree Tripathi
- PhD Student
Mathieu Brochet
- unknown
I studied Agronomy at the National Higher Agronomic School (France) and at McGill University (Canada). After completion of my undergraduate training I joined the Genomics of Microbial Pathogens unit at the Pasteur Institute (France) and took up a project centring on the evolution of Group B Streptococcus, the leading cause of neonatal infections. After completing my PhD work, I undertook a postdoctoral position with Oliver Billker at the Wellcome Trust Sanger Institute.
My current work aims at identifying the molecular links between essential signalling pathways and the molecular motor which allows Plasmodium parasites to successfully invade host cells. Specifically, I am interested in identifying and characterising the cGMP- and calcium-dependent signalling pathways. This involves the use of Plasmodium specific genetic and chemical genetic approaches, comparative phosphoproteomic and quantitative phenotyping of motility behaviour.
A scalable pipeline for highly effective genetic modification of a malaria parasite.
The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei.
Funded by: Medical Research Council: G0501670, G0501670(76331); Wellcome Trust: 089085, WT089085/Z/09/Z
Nature methods 2011;8;12;1078-82
PUBMED: 22020067; PMC: 3431185; DOI: 10.1038/nmeth.1742
Population structure of human isolates of Streptococcus agalactiae from Dakar and Bangui.
Institut Pasteur, Unité de Génétique des Génomes Bactériens, CNRS URA 2171, 28 Rue du Dr Roux, 75724 Paris Cedex 15, France.
Multilocus sequence types of 163 human Streptococcus agalactiae strains isolated in Bangui and Dakar were analyzed. We identified local specificities in the distribution of sequence types and capsular serotypes. However, the overall population structure is similar to that in the United States and Europe, suggesting that few specific clones colonize humans.
Journal of clinical microbiology 2009;47;3;800-3
Atypical association of DDE transposition with conjugation specifies a new family of mobile elements.
Institut Pasteur, Unité de Génétique des Génomes Bactériens, CNRS URA 2171, France.
We describe in Streptococcus agalactiae an atypical family of conjugative transposons named TnGBSs which associates DDE transposition and conjugation. We present evidence that the transposition of TnGBS2, the prototype of this family, is catalysed by a new class of DDE transposases that are widespread in Gram-positive bacteria. Remarkably, transposition occurs in intergenic regions, 15 or 16 bp upstream the -35 sequence of promoters, minimizing the burden on the host cell and suggesting an association between transcription and transposition. Transposition catalyses the formation of a circular intermediate that is substrate for subsequent conjugative intercellular transfer. Conjugation is initiated at an origin of transfer by a transposon-encoded relaxase. Whereas all integrative and conjugative elements described so far encode a phage-related integrase, TnGBS2 is the first example of conjugative transposon whose recombination is mediated by a DDE transposase. The combination of DDE transposition with conjugation implies recombination constraints linked to the physical separation of donor and recipient molecules.
Molecular microbiology 2009;71;4;948-59
PUBMED: 19183283; DOI: 10.1111/j.1365-2958.2008.06579.x
Shaping a bacterial genome by large chromosomal replacements, the evolutionary history of Streptococcus agalactiae.
Institut Pasteur, Unité de Génétique des Génomes Bactériens, Centre National de la Recherche Scientifique URA 2171.
Bacterial populations are subject to complex processes of diversification that involve mutation and horizontal DNA transfer mediated by transformation, transduction, or conjugation. Tracing the evolutionary events leading to genetic changes allows us to infer the history of a microbe. Here, we combine experimental and in silico approaches to explore the forces that drive the genome dynamics of Streptococcus agalactiae, the leading cause of neonatal infections. We demonstrate that large DNA segments of up to 334 kb of the chromosome of S. agalactiae can be transferred through conjugation from multiple initiation sites. Consistently, a genome-wide map analysis of nucleotide polymorphisms among eight human isolates demonstrated that each chromosome is a mosaic of large chromosomal fragments from different ancestors suggesting that large DNA exchanges have contributed to the genome dynamics in the natural population. The analysis of the resulting genetic flux led us to propose a model for the evolutionary history of this species in which clonal complexes of clinical importance derived from a single clone that evolved by exchanging large chromosomal regions with more distantly related strains. The emergence of this clone could be linked to selective sweeps associated with the reduction of genetic diversity in three regions within a large panel of human isolates. Up to now sex in bacteria has been assumed to involve mainly small regions; our results define S. agalactiae as an alternative paradigm in the study of bacterial evolution.
Proceedings of the National Academy of Sciences of the United States of America 2008;105;41;15961-6
PUBMED: 18832470; PMC: 2572952; DOI: 10.1073/pnas.0803654105
Integrative conjugative elements and related elements are major contributors to the genome diversity of Streptococcus agalactiae.
Unité de Génétique des Génomes Bactériens-URA CNRS2171, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris cedex 15, France.
Thirty-five putative integrative conjugative elements and related elements were identified at 15 locations in the eight sequenced genomes of Streptococcus agalactiae. Twelve are composite, likely resulting from site-specific accretions. Circular forms were detected for five elements. Macroarray analysis confirmed their high plasticity and wide distribution in S. agalactiae.
Journal of bacteriology 2008;190;20;6913-7
A naturally occurring gene amplification leading to sulfonamide and trimethoprim resistance in Streptococcus agalactiae.
Unité de Génomique des Microorganismes Pathogènes, Institut Pasteur, 28 Rue du Dr. Roux, 75724 Paris cedex 15, France.
Gene amplifications have been detected as a transitory phenomenon in bacterial cultures. They are predicted to contribute to rapid adaptation by simultaneously increasing the expression of genes clustered on the chromosome. However, genome amplifications have rarely been described in natural isolates. Through DNA array analysis, we have identified two Streptococcus agalactiae strains carrying tandem genome amplifications: a fourfold amplification of 13.5 kb and a duplication of 92 kb. Both amplifications were located close to the terminus of replication and originated independently from any long repeated sequence. They probably arose in the human host and showed different stabilities, the 13.5-kb amplification being lost at a frequency of 0.003 per generation and the 92-kb tandem duplication at a frequency of 0.035 per generation. The 13.5-kb tandem amplification carried the five genes required for dihydrofolate biosynthesis and led to both trimethoprim (TMP) and sulfonamide (SU) resistance. Resistance to SU probably resulted from the increased synthesis of dihydropteroate synthase, the target of this antibiotic, whereas the amplification of the whole pathway was responsible for TMP resistance. This revealed a new mechanism of resistance to TMP involving an increased dihydrofolate biosynthesis. This is, to our knowledge, the first reported case of naturally occurring antibiotic resistance resulting from genome amplification in bacteria. The low stability of DNA segment amplifications suggests that their role in antibiotic resistance might have been underestimated.
Journal of bacteriology 2008;190;2;672-80
Genomic diversity and evolution within the species Streptococcus agalactiae.
Unité de Génomique des Microorganismes Pathogènes-URA CNRS 2171, Institut Pasteur, 25-28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Streptococcus agalactiae is a leading cause of invasive infections in neonates, and responsible for bovine mastitis. It is also a commensal bacterium adapted to asymptomatic colonization of the mammalian gut and of the genitourinary tract. Here, we report the analysis of a collection of 75 strains of human and animal origin by using serotyping, multilocus sequence typing, whole genome DNA-array hybridizations and sequence comparison of putatively virulence-associated loci. Although the most variable parts of the genome are the previously predicted genomic islands, significant genetic variations were present in the genome backbone. Evolution within genes encoding surface and secreted proteins and those involved in the biosynthesis of different capsular types is mainly due to recombination events leading to the replacement of a locus of several genes or to the allelic exchange of the internal part of a gene. These two processes, which led to a broad diversity of surface protein patterns, are probably involved in the diversity of interactions with the host and its immune system. According to gene content comparisons and phylogeny, recent gene replacements by horizontal gene transfer may occur but are rare events. Although specific gene patterns, with respect to the origin of the strains and the epidemiological characteristics, were not identified, we show that the recently described hypervirulent ST-17 lineage is a homogeneous group. The study highlights for the first time that this lineage contains a specific and conserved set of surface proteins, probably accounting for its high capacity to cause infections in newborns.
Microbes and infection / Institut Pasteur 2006;8;5;1227-43
PUBMED: 16529966; DOI: 10.1016/j.micinf.2005.11.010
Burcu Bronner Anar
- Advanced Research Assistant
I completed my B.Sc. and M.Sc. in the Biology department at METU (Middle East Technical University) in Ankara, Turkey between 1990 and 1998. I then worked as a senior research assistant at the Erasmus University in Rotterdam and at the VU (Vrije Universiteit) in Amsterdam (1999-2007), on the characterization of molecular mechanisms involved in the formation of insoluble aggregates in neurodegenerative diseases.
I joined the Sanger Malaria programme in 2008. I have been involved since the beginning in the adaptation and optimisation of a recombineering based method for the generation of gene targeting vectors for the rodent malaria parasite Plasmodium berghei. This technology now forms the basis of the gene targeting vector pipeline that underpins the Plasmodium genetic modification (PlasmoGEM) project (http://plasmogem.sanger.ac.uk/). Within the PlasmoGEM team I continue to work on technology development, vector production and Plasmodium genomic library generation.
Recombination-mediated genetic engineering of Plasmodium berghei DNA.
Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
DNA of Plasmodium berghei is difficult to manipulate in Escherichia coli by conventional restriction and ligation methods due to its high content of adenine and thymine (AT) nucleotides. This limits our ability to clone large genes and to generate complex vectors for modifying the parasite genome. We here describe a protocol for using lambda Red recombinase to modify inserts of a P. berghei genomic DNA library constructed in a linear, low-copy, phage-derived vector. The method uses primer extensions of 50 bp, which provide sufficient homology for an antibiotic resistance marker to recombine efficiently with a P. berghei genomic DNA insert in E. coli. In a subsequent in vitro Gateway reaction the bacterial marker is replaced with a cassette for selection in P. berghei. The insert is then released and used for transfection. The basic techniques we describe here can be adapted to generate highly efficient vectors for gene deletion, tagging, targeted mutagenesis, or genetic complementation with larger genomic regions.
Funded by: Medical Research Council: G0501670
Methods in molecular biology (Clifton, N.J.) 2013;923;127-38
A scalable pipeline for highly effective genetic modification of a malaria parasite.
The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei.
Funded by: Medical Research Council: G0501670, G0501670(76331); Wellcome Trust: 089085, WT089085/Z/09/Z
Nature methods 2011;8;12;1078-82
PUBMED: 22020067; PMC: 3431185; DOI: 10.1038/nmeth.1742
Ellen Bushell
email@example.com Senior Staff Scientist
I graduated from Cardiff University in 2004, with a BSc Hons in Applied Biology and a year in industry at AstraZeneca. After my first degree I entered the Wellcome Trust 4-year PhD Infection programme at Imperial College, London. Upon completing my MRes year I joined the Kafatos / Christophides lab to pursue a PhD working on the transmission stages of Plasmodium berghei. In 2009, remaining at Imperial College, I took up a position as a postdoctoral fellow funded by the TransMalBloc consortium. My postdoc focused on monitoring transcriptional regulation of Plasmodium falciparum transmission in the field, in Burkina Faso, West Africa.
In 2011 I joined the Sanger Malaria Programme as a staff scientist. My primary research interest is the development of tools for high throughput reverse genetic analysis of the malaria parasite. I function as the project manager for a new exciting initiative; the Plasmodium genetic modification (PlasmoGEM) project (http://plasmogem.sanger.ac.uk/), and in this role I am part of the team that is working towards producing genome-wide, open access Plasmodium vector resources.
Genetic susceptibility to the delayed sequelae of neonatal respiratory syncytial virus infection is MHC dependent.
Department of Respiratory Medicine, Centre for Respiratory Infection, National Heart and Lung Institute, Imperial College London, London, United Kingdom.
Respiratory syncytial virus (RSV) is a major cause of respiratory morbidity, resulting in hospitalization for bronchiolitis in some infected infants that is associated with wheeze in later life. Genetic factors are known to affect the severity of the sequelae after RSV infection, but the complexity of the temporal and genetic effects makes it difficult to analyze this response in studies in man. Therefore, we developed a murine genetic model to analyze the sequelae occurring after RSV infection in early life. Haplotype-based genetic analysis of interstrain differences in severity identified the MHC as an important genetic determinant. This was confirmed by analysis of responses in congenic mice with different MHC haplotypes. We also found that susceptible strains had high CD8 levels during secondary infection. Analysis of first filial generation, second filial generation, and back-cross progeny produced by intercrossing resistant (H-2(k), C3H/HeN) and sensitive (H-2(b), BALB/c) strains indicated that susceptibility to sequelae after RSV infection was dominantly inherited but also segregated in a non-MHC-dependent manner. Thus, MHC haplotype and its effect on CD8 cell response is an important determinant of the outcome of neonatal RSV infection.
Funded by: Medical Research Council; NIGMS NIH HHS: 1 R01 GM068885-01A1; Wellcome Trust: 071381/Z/03/Z
Journal of immunology (Baltimore, Md. : 1950) 2010;185;9;5384-91
PUBMED: 20921522; DOI: 10.4049/jimmunol.1001594
Paternal effect of the nuclear formin-like protein MISFIT on Plasmodium development in the mosquito vector.
Department of Life Sciences, Imperial College London, London, UK.
Malaria parasites must undergo sexual and sporogonic development in mosquitoes before they can infect their vertebrate hosts. We report the discovery and characterization of MISFIT, the first protein with paternal effect on the development of the rodent malaria parasite Plasmodium berghei in Anopheles mosquitoes. MISFIT is expressed in male gametocytes and localizes to the nuclei of male gametocytes, zygotes and ookinetes. Gene disruption results in mutant ookinetes with reduced genome content, microneme defects and altered transcriptional profiles of putative cell cycle regulators, which yet successfully invade the mosquito midgut. However, developmental arrest ensues during the ookinete transformation to oocysts leading to malaria transmission blockade. Genetic crosses between misfit mutant parasites and parasites that are either male or female gamete deficient reveal a strict requirement for a male misfit allele. MISFIT belongs to the family of formin-like proteins, which are known regulators of the dynamic remodeling of actin and microtubule networks. Our data identify the ookinete-to-oocyst transition as a critical cell cycle checkpoint in Plasmodium development and lead us to hypothesize that MISFIT may be a regulator of cell cycle progression. This study offers a new perspective for understanding the male contribution to malaria parasite development in the mosquito vector.
Funded by: Wellcome Trust: 075176/Z/04/Z, GR077229/Z/05/Z
PLoS pathogens 2009;5;8;e1000539
PUBMED: 19662167; PMC: 2715856; DOI: 10.1371/journal.ppat.1000539
Reverse genetics screen identifies six proteins important for malaria development in the mosquito.
Division of Cell and Molecular Biology, Imperial College London, London SW7 2AZ, UK. firstname.lastname@example.com
Transmission from the vertebrate host to the mosquito vector represents a major population bottleneck in the malaria life cycle that can successfully be targeted by intervention strategies. However, to date only about 25 parasite proteins expressed during this critical phase have been functionally analysed by gene disruption. We describe the first systematic, larger scale generation and phenotypic analysis of Plasmodium berghei knockout (KO) lines, characterizing 20 genes encoding putatively secreted proteins expressed by the ookinete, the parasite stage responsible for invasion of the mosquito midgut. Of 12 KO lines that were generated, six showed significant reductions in parasite numbers during development in the mosquito, resulting in a block in transmission of five KOs. While expression data, time point of essential function and mutant phenotype correlate well in three KOs defective in midgut invasion, in three KOs that fail at sporulation, maternal inheritance of the mutant phenotype suggests that essential function occurs during ookinete formation and thus precedes morphological abnormalities by several days.
Funded by: Wellcome Trust
Molecular microbiology 2008;70;1;209-20
PUBMED: 18761621; PMC: 2658712; DOI: 10.1111/j.1365-2958.2008.06407.x
The developmental migration of Plasmodium in mosquitoes.
Division of Cell and Molecular Biology, Faculty of Natural Sciences, Imperial College London, London, SW7 2AZ, UK. firstname.lastname@example.com
Migration of the protozoan parasite Plasmodium through the mosquito is a complex and delicate process, the outcome of which determines the success of malaria transmission. The mosquito is not simply the vector of Plasmodium but, in terms of the life cycle, its definitive host: there, the parasite undergoes its sexual development, which results in colonization of the mosquito salivary glands. Two of the parasite's developmental stages in the mosquito, the ookinete and the sporozoite, are invasive and depend on gliding motility to access, penetrate and traverse their host cells. Recent advances in the field have included the identification of numerous Plasmodium molecules that are essential for parasite migration in the mosquito vector.
Funded by: NIAID NIH HHS: AI044200-08
Current opinion in genetics & development 2006;16;4;384-91
PUBMED: 16793259; DOI: 10.1016/j.gde.2006.06.012
Tetracycline-inducible gene regulation in mycobacteria.
Department of Infectious Diseases and Microbiology, Centre for Molecular Microbiology and Infection, Imperial College London South Kensington campus, London SW7 2AZ, UK.
A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml(-1) of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells.
Funded by: Medical Research Council: MC_U117581288
Nucleic acids research 2005;33;2;e22
PUBMED: 15687380; PMC: 548381; DOI: 10.1093/nar/gni023
Gareth Girling
- Advanced Research Assistant
I graduated from the University of Essex with a BSc in Cell and Molecular Biology in 2006. Following this I worked for four years at GlaxoSmithKline in the Screening and Compound Profiling department, where I was predominantly involved in generating high throughput structure-activity relationship data for small molecule drug candidates against kinase targets.
I joined the Sanger Malaria Programme in 2011. I work on the development and day-to-day running of the high-throughput Plasmodium gene targeting vector pipeline that form the basis of the Plasmodium genetic modification (PlasmoGEM) project (http://plasmogem.sanger.ac.uk/). The aim of PlasmoGEM is to generate genome-wide libraries of gene knockout and tagging vectors for Plasmodium, available for use by the wider research community. Our current focus is on the rodent malaria species Plasmodium berghei.
Stefano Iantorno
- PhD Student
Stefano graduated from the University of California, Berkeley in 2010 with a B.A. in Integrative Biology and a Minor in Rhetoric. He spent a semester abroad at the Instituto Monteverde, Costa Rica, studying Tropical Biology and Conservation, and worked at UCSF as Staff Research Associate.
As a PhD student in the NIH-Oxford-Cambridge Scholars Program, Stefano is pursuing a research project with the Sanger Institute Malaria Programme and the Laboratory of Malaria and Vector Research at the US National Institutes of Health, supervised by Dominic Kwiatkowski, Oliver Billker and Carolina Barillas-Mury.
Stefano's research focuses on mapping genes in the Plasmodium genome that are involved in vector-parasite interactions in several vector species, high-throughput whole-genome sequencing of parasite DNA from infected mosquitoes and population-level analyses of potential loci of interest.
Kasia Modrzynska
firstname.lastname@example.com Staff Scientist
Ruddy Montandon
- Postdoctoral Fellow
Frank Schwach
firstname.lastname@example.com Senior Computational Biologist
I obtained a PhD in plant virology at the University of Hamburg (Germany) in 2002 before joining the lab of David Baulcombe at the Sainsbury Laboratory in Norwich (UK), working on the mechanisms of small-RNA-mediated anti-viral gene silencing. With the advent of next-generation sequencing, I shifted my focus to the computational analysis of high-throughput sequence data. I continued this work at the University of East Anglia, developing a collection of web-based tools for the analysis of large-scale small RNA data. I joined the Billker lab in December 2010.
My main project is the development of the software tools and the web portal (PLasmoGEM) for the high-throughput Malaria gene targeting pipeline and the computational data analysis to support the project. In addition, I provide bioinformatics support for various projects in the lab, including transcriptomics and proteomics assays.
PfSET10, a Plasmodium falciparum methyltransferase, maintains the active var gene in a poised state during parasite division.
The Walter and Eliza Hall Institute for Medical Research, Melbourne, Victoria, Australia.
A major virulence factor of the malaria parasite Plasmodium falciparum is erythrocyte membrane protein 1 (PfEMP1), a variant protein expressed on the infected erythrocyte surface. PfEMP1 is responsible for adherence of infected erythrocytes to the endothelium and plays an important role in pathogenesis. Mutually exclusive transcription and switched expression of one of 60 var genes encoding PfEMP1 in each parasite genome provides a mechanism for antigenic variation. We report the identification of a parasite protein, designated PfSET10, which localizes exclusively to the perinuclear active var gene expression site. PfSET10 is a histone 3 lysine 4 methyltransferase required to maintain the active var gene in a poised state during division for reactivation in daughter parasites, and as such is required for P. falciparum antigenic variation. PfSET10 likely maintains the transcriptionally permissive chromatin environment of the active var promoter and thus retains memory for heritable transmission of epigenetic information during parasite division.
Cell host & microbe 2012;11;1;7-18
PUBMED: 22264509; DOI: 10.1016/j.chom.2011.11.011
A scalable pipeline for highly effective genetic modification of a malaria parasite.
The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.
In malaria parasites, the systematic experimental validation of drug and vaccine targets by reverse genetics is constrained by the inefficiency of homologous recombination and by the difficulty of manipulating adenine and thymine (A+T)-rich DNA of most Plasmodium species in Escherichia coli. We overcame these roadblocks by creating a high-integrity library of Plasmodium berghei genomic DNA (>77% A+T content) in a bacteriophage N15-based vector that can be modified efficiently using the lambda Red method of recombineering. We built a pipeline for generating P. berghei genetic modification vectors at genome scale in serial liquid cultures on 96-well plates. Vectors have long homology arms, which increase recombination frequency up to tenfold over conventional designs. The feasibility of efficient genetic modification at scale will stimulate collaborative, genome-wide knockout and tagging programs for P. berghei.
Funded by: Medical Research Council: G0501670, G0501670(76331); Wellcome Trust: 089085, WT089085/Z/09/Z
Nature methods 2011;8;12;1078-82
PUBMED: 22020067; PMC: 3431185; DOI: 10.1038/nmeth.1742
Profiling of short RNAs during fleshy fruit development reveals stage-specific sRNAome expression patterns.
School of Computing Sciences, University of East Anglia, Norwich, UK.
Plants feature a particularly diverse population of short (s)RNAs, the central component of all RNA silencing pathways. Next generation sequencing techniques enable deeper insights into this complex and highly conserved mechanism and allow identification and quantification of sRNAs. We employed deep sequencing to monitor the sRNAome of developing tomato fruits covering the period between closed flowers and ripened fruits by profiling sRNAs at 10 time-points. It is known that microRNAs (miRNAs) play an important role in development but very little information is available about the majority of sRNAs that are not miRNAs. Here we show distinctive patterns of sRNA expression that often coincide with stages of the developmental process such as flowering, early and late fruit maturation. Moreover, thousands of non-miRNA sRNAs are differentially expressed during fruit development and ripening. Some of these differentially expressed sRNAs derived from transposons but many derive from protein coding genes or regions that show homology to protein coding genes, several of which are known to play a role in flower and fruit development. These findings raise the possibility of a regulative role of these sRNAs during fruit onset and maturation in a crop species. We also identified six new miRNAs and experimentally validated two target mRNAs. These two mRNAs are targeted by the same miRNA but do not belong to the same gene family, which is rare for plant miRNAs. Expression pattern and putative function of these targets indicate a possible role in glutamate accumulation, which contributes to establishing the taste of the fruit.
Funded by: Biotechnology and Biological Sciences Research Council: BB/G008078/1, BB/I00016X/1
The Plant journal : for cell and molecular biology 2011;67;2;232-46
PUBMED: 21443685; DOI: 10.1111/j.1365-313X.2011.04586.x
Deciphering the diversity of small RNAs in plants: the long and short of it.
School of Computing Sciences, University of East Anglia, Norwich, UK. firstname.lastname@example.com
RNA silencing is a complex and highly conserved regulatory mechanism that is now known to be involved in such diverse processes as development, pathogen control, genome maintenance and response to environmental changes. Since its recent discovery, RNA silencing has become a fast moving key area of research in plant and animal molecular biology. Research in this field has greatly profited from recent developments in novel sequencing technologies that allow massive parallel sequencing of small RNA (sRNA) molecules, the key players of all RNA silencing phenomena. As researchers are beginning to decipher the complexity of RNA silencing, novel methodologies have to be developed to make sense of the large amounts of data that are currently being generated. In this review we present an overview of RNA silencing pathways in plants and the current challenges in analysing sRNA data, with a special focus on computational approaches.
Funded by: Biotechnology and Biological Sciences Research Council: BB/E004091/1
Briefings in functional genomics & proteomics 2009;8;6;472-81
PUBMED: 19641088; DOI: 10.1093/bfgp/elp024
A toolkit for analysing large-scale plant small RNA datasets.
School of Computing Sciences, School of Biological Sciences, University of East Anglia, Norwich NR47TJ, UK.
Unlabelled: Recent developments in high-throughput sequencing technologies have generated considerable demand for tools to analyse large datasets of small RNA sequences. Here, we describe a suite of web-based tools for processing plant small RNA datasets. Our tools can be used to identify micro RNAs and their targets, compare expression levels in sRNA loci, and find putative trans-acting siRNA loci.
Availability: The tools are freely available for use at http://srna-tools.cmp.uea.ac.uk.
Funded by: Biotechnology and Biological Sciences Research Council: BB/E004091/1
Bioinformatics (Oxford, England) 2008;24;19;2252-3
PUBMED: 18713789; DOI: 10.1093/bioinformatics/btn428
PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis.
Sainsbury Laboratory, Norwich NR4 7UH, United Kingdom.
DNA-dependent RNA polymerase (Pol)IV in Arabidopsis exists in two isoforms (PolIVa and PolIVb), with NRPD1a and NRPD1b as their respective largest subunits. Both isoforms are implicated in production and activity of siRNAs and in RNA-directed DNA methylation (RdDM). Deep sequence analysis of siRNAs in WT Arabidopsis flowers and in nrpd1a and nrpd1b mutants identified >4,200 loci producing siRNAs in a PolIV-dependent manner, with PolIVb reinforcing siRNA production by PolIVa. Transposable element identity and pericentromeric localization are both features that predispose a locus for siRNA production via PolIV proteins and determine the extent to which siRNA production relies on PolIVb. Detailed analysis of DNA methylation at PolIV-dependent loci revealed unexpected deviations from the previously noted association of PolIVb-dependent siRNA production and RdDM. Notably, PolIVb functions independently in DNA methylation and siRNA generation. Additionally, we have uncovered siRNA-directed loss of DNA methylation, a process requiring both PolIV isoforms. From these findings, we infer that the role of PolIVb in siRNA production is secondary to a role in chromatin modification and is influenced by chromatin context.
Proceedings of the National Academy of Sciences of the United States of America 2008;105;8;3145-50
PUBMED: 18287047; PMC: 2268599; DOI: 10.1073/pnas.0709632105
miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii.
Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK.
MicroRNAs (miRNAs) in eukaryotes guide post-transcriptional regulation by means of targeted RNA degradation and translational arrest. They are released by a Dicer nuclease as a 21-24-nucleotide RNA duplex from a precursor in which an imperfectly matched inverted repeat forms a partly double-stranded region. One of the two strands is then recruited by an Argonaute nuclease that is the effector protein of the silencing mechanism. Short interfering RNAs (siRNAs), which are similar to miRNAs, are also produced by Dicer but the precursors are perfectly double-stranded RNA. These siRNAs guide post-transcriptional regulation, as with miRNAs, and epigenetic genome modification. Diverse eukaryotes including fungi, plants, protozoans and metazoans produce siRNAs but, until now, miRNAs have not been described in unicellular organisms and it has been suggested that they evolved together with multicellularity in separate plant and animal lineages. Here we show that the unicellular alga Chlamydomonas reinhardtii contains miRNAs, putative evolutionary precursors of miRNAs and species of siRNAs resembling those in higher plants. The common features of miRNAs and siRNAs in an alga and in higher plants indicate that complex RNA-silencing systems evolved before multicellularity and were a feature of primitive eukaryotic cells.
Nature 2007;447;7148;1126-9
PUBMED: 17538623; DOI: 10.1038/nature05903
An RNA-dependent RNA polymerase prevents meristem invasion by potato virus X and is required for the activity but not the production of a systemic silencing signal.
Sainsbury Laboratory, Norwich NR4 7UH, United Kingdom.
One of the functions of RNA silencing in plants is antiviral defense. A hallmark of RNA silencing is spreading of the silenced state through the plant. Little is known about the nature of the systemic silencing signal and the proteins required for its production, transport, and reception in plant tissues. Here, we show that the RNA-dependent RNA polymerase RDR6 in Nicotiana benthamiana is involved in defense against potato virus X at the level of systemic spreading and in exclusion of the virus from the apical growing point. It has no effect on primary replication and cell-to-cell movement of the virus and does not contribute significantly to the formation of virus-derived small interfering (si) RNA in a fully established potato virus X infection. In grafting experiments, the RDR6 homolog was required for the ability of a cell to respond to, but not to produce or translocate, the systemic silencing signal. Taking these findings together, we suggest a model of virus defense in which RDR6 uses incoming silencing signal to generate double-stranded RNA precursors of secondary siRNA. According to this idea, the secondary siRNAs mediate RNA silencing as an immediate response that slows down the systemic spreading of the virus into the growing point and newly emerging leaves.
Plant physiology 2005;138;4;1842-52
PUBMED: 16040651; PMC: 1183376; DOI: 10.1104/pp.105.063537
Jaishree Tripathi
- PhD Student
Jaishree obtained a BSc in Life Sciences majoring in Biochemistry from St. Xavier's College, Mumbai, India, followed by an MSc in Biotechnology from the University of Hyderabad, India, where she worked with Dr. Mrinal K. Bhattacharya on the Role of PfORC1 in Sir protein mediated gene silencing in P. falciparum.
Jaishree is a joint EVIMalaR PhD student with Prof. Maria M. Mota. Her aim is to develop in vitro differentiated hepatocytes from human induced pluripotent stem cells as a model to get at the functional impact of natural variation in humans on malaria liver infection.


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Finding a Real Estate Agent in Astoria
Coldwell Banker Real Estate Agents near Astoria, South Dakota Can Turn Your Dream of Homeownership into a Reality
While there are not currently any Coldwell Banker real estate agents in Astoria, rest assured that there are Coldwell Banker agents from neighboring cities and towns that are committed to delivering superior home buying and selling services to the Astoria community. Each real estate agent servicing Astoria, SD is equipped with the local knowledge and sophisticated real estate tools to help you sell your existing home or buy the Astoria home of your dreams.
Start your search today by viewing Astoria real estate listings.

subset: commoncrawl


text:
Produce thumbnails the easy way,
quickly and for free!
Easy Thumbnails is a popular free Windows utility for creating accurate thumbnail images and scaled-down/up copies from a wide range of popular picture formats. An elegant interface makes it a snap to find your images and select them for processing individually, in groups, or in whole folders, using a simple file selector and built-in image viewer. You can use slider controls to rotate images and adjust their contrast, brightness, sharpness and quality, and check out the results with the built-in viewer.
Thumbnails can be created in any existing folder or a new folder, and you can identify them clearly by adding a prefix or suffix to their filename. If you're an image-processing enthusiast, you'll enjoy having a choice of eleven resampling filters for the best possible results. You can convert small batches of images inside Windows Explorer from the File menu or with a right-click of the mouse, display thumbnails in your browser, and even take advantage of command-line switches to generate thumbnails seamlessly from other programs. You can also save your thumbnails to a Web page using a HTML template that you can easily customize to create your own layouts, and there's helpful documentation that includes full instructions and some useful tips.
Easy Thumbnails is a genuine freeware product that will not annoy you with advertising, intrusive spyware components or nag screens. It comes from the developers of the award-winning NoteTab text editors.
This page has been translated into Spanish by Maria Ramos from Webhostinghub.com/support/edu.
Aid4Mail, NoteTab, Mailbag Assistant, Easy Imager, Album Express, and Easy Thumbnails
are all trademarks licensed to Fookes Software Ltd.
Fookes is a registered trademark of Fookes Holding Ltd.
Windows is a trademark of Microsoft Corporation registered in the U.S. and other countries.
All other trademarks and service marks, both marked and not marked, are the property of their respective owners.
Last updated on October 8, 2009


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At Guy's Cross Farm you will find a charming self-contained luxury cottage located in quiet rural surroundings just 20 minutes from the attractions of Canberra and 10 minutes from Queanbeyan. Pets welcome by arrangement. Guy's Cross Cottage accommodates two people in large king single beds with room for two additional guests, all the linen is provided. Amenities include; full sized tennis court, BBQ, fully appointed kitchen, bathroom and undercover parking. All the normal facilities such as TV, video, DVD, iron, hair dryer, etc are also provided. Guests are welcome to take a farm walk through our olive groves, enjoy the panoramic views and visit our beef cattle and farm nature reserves. Kangaroos will usually be encountered on an evening stroll. For the energetic, a game of tennis may appeal. We can provide racquets and balls. Guests are also welcome to any fresh fruit or vegetables from our gardens and orchard whenever they are in season. Guys cross is an ideal base for those wishing to range further afield in the district and from extensive experience we can provide ideas and advice on country drives, bushwalking and bird watching.
Canberra, A.C.T., Australia (CBR) 10 miles
1. Head southeast on Terminal Cct toward Ulinga Pl 84 m 2. Turn right at Ulinga Pl 12 m 3. Take the 1st left onto Terminal Cct 0.2 km 4. Slight left at Brindabella Cct 63 m 5. Turn right to stay on Brindabella Cct 0.8 km 6. Turn right to stay on Brindabella Cct 0.1 km 7. Take the 1st left onto Pialligo Ave 4.7 km 8. Slight right at Yass Rd Entering New South Wales 1.8 km 9. Turn left at Thurralilly St 0.4 km 10. Take the 2nd left onto Kings Hwy 2.5 km 11. Turn right at Captains Flat Rd 4.2 km 12. Turn left Destination will be on the left
BBQ grill
Hairdryer, Ironing Board, TV
Tim Booth
BedandBreakfast.com Diamond Collection


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EDMONTON - The Edmonton Economic Development Corporation has announced plans for a 1,000-acre Sturgeon County industrial park that would include a facility to make liquefied natural gas for export to China.
EEDC said Monday it has signed a memorandum of understanding with Gascana AB Energy Ltd. and Gascana-Sino Industrial Park Administrative Committee Inc. on a project called the Sino Canada Natural Gas Development Initiative.
It would focus on natural-gas related industries including refining, upgrading, technology research and manufacturing, the partners said.
The Sturgeon County site would include LNG production, up to five facilities to extract chemicals from natural gas, equipment manufacturing and professional services. A related business incubator, backed by a $30-million equity fund, would open in Edmonton.
Brad Ferguson, EEDC's president and CEO, said the project has come together over the past 10 months. More work lies ahead in terms of market assessment and regulatory approvals, he said.
"That's why I guess I'm not clicking my heels too much today," Ferguson said. "Over the next year it all starts to come together in terms of the viability.
"These folks are very committed, that's all I can tell you. But until there are shovels in the ground and there's things flowing, then any deal ... can fall off track."
Preliminary details of the LNG plant were made public last week in a news release from Calgary junior oil and gas firm Petrox Resources Corp.
The release described Gascana as a new joint venture between Petrox, Qingdao Sinoenergy Corp. of China and Asiafic Clean Energy Ltd., a Hong Kong subsidiary of Qingdao Sinoenergy.
Petrox said Gascana plans to build an LNG plant in Alberta with daily production capacity of 2,950 tonnes. The release didn't specify a location but Ferguson confirmed it's the same plant being discussed for Sturgeon County.
Gascana hasn't said how it would export its products but Ferguson said the LNG could be moved to the West Coast by rail, pipeline or truck.
The industrial park would be built in three phases over five years on land near Highway 825 and Range Road 222 in the Industrial Heartland.
Total investment would be $4 billion, EEDC said. When complete, it would achieve $5 billion in annual production and provide more than 2,000 permanent jobs, EEDC said.
EEDC will continue to work with the project as it moves forward, Ferguson said.
The Sino Edmonton incubator will provide help to local businesses that are designed from "Day 1" to export goods to China, he said.
Jordan Rumohr, manager of economic development for Sturgeon County, said county officials are aware of the industrial park proposal and the work EEDC has done.
"We're kind of light on details, on specifics," Rumohr said. "We've been aware of them for a little while now but we're excited about the opportunity to work with them."
In its June 19 release, Petrox said it would have a 10 per cent equity interest in the LNG plant. It would implement the project and obtain regulatory approvals. Asiafic would hold the remaining 90 per cent interest. It would secure the upstream natural gas supply and purchase the land. Gascana would be responsible for engineering and construction.
On April 1, the National Energy Board authorized Gascana Inc. to export liquefied natural gas for a period of two years beginning April 2.
The Alberta Energy Regulator has not received any applications related to the project, spokesman Darin Barter said.


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Sonic Unleashed Wii Walkthrough Part 19 - Movies
Press the button below if you liked this video! Thank you! =D
1,615 views
Posted By supersonicfan 2011-07-29T12:54:53
Time to head back to Spagonia for some daytime Sonic speed! Honestly, this is probably my worst run on this level ever, so sorry! Sonic just seemed to like face-planting the walls this time!
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Little Red Riding Hood went to her grandma's house and found her laying in bed.
Little Red Riding Hood said, ''Grandma, what big eyes you have!''
Grandma: ''The better to see you with, my dear.''
Little Red Riding Hood: ''Grandma, what big ears you have!''
Grandma: "The better to hear you with, my dear.''
Little Red Riding Hood: ''Grandma, what a big mouth you have!''
Grandma: ''Of course I do, have you seen Grandpa's d**k?!?''
The web series from CC:Studios will return in television form.
Unlimited streams and downloads of the best stand-up specials for just $5.
Visit our CC Live page for live comedy events near you.


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DALLAS – Cowboys linebacker Sean Lee was a featured speaker at the recent Texas AgriLife Extension Service and Fuel Up to Play 60 Partnership kickoff, which took place at the Texas AgriLife Research and Extension Center, 17360 Coit Road in Dallas.
Fuel Up to Play 60 is an in-school nutrition and physical activity program launched by the National Dairy Council, Dairy MAX and the National Football League, in collaboration with U.S. Department of Agriculture. It encourages young people to consume nutrient-rich foods, including low-fat and fat-free dairy foods, fruits, vegetables and whole grains, and to get at least 60 minutes of daily physical activity.
"Fuel Up to Play 60 is a great initiative to get kids interested in exercise and nutrition," said Lee, who has been a program spokesman and supporter since 2010. "If you get kids interested in a healthy lifestyle, it will help them in whatever career or endeavor that may have by making them feel better, have more energy and have better concentration."
The kickoff, which included speakers' presentations, a press conference, an exercise warm-up with more than 30 area 4-H club members and a 5K walk/run led by Lee, officially welcomed AgriLife Extension to the program. It also introduced AgriLife Extension's plans to support and promote Fuel Up to Play 60 statewide, beginning with their multi-county East Region, which includes Dallas and Tarrant counties.
"I had seen the Play 60 commercials on TV and was really excited that 4-H would have the opportunity to play a part in the program," said Catlyn Wold, 17, a senior at Wills Point High School.
Wold, who participated in program activities, serves as president of the Kaufman County 4-H Youth Council. Texas 4-H and Youth Development is the statewide youth program of AgriLife Extension.
"The whole event was amazing in every aspect," she said. "It was great to meet Sean Lee and to hear from Mr. Ramsey, who is a dairyman and one of our biggest area 4-H supporters. I'm also glad the NFL is behind this program, because the players are our heroes."
4-H club members at the event also represented Collin and Denton counties.
In addition to Lee and 4-H club members, event participants included Shelly Sturges, district aide to Sen. John Carona; Katy Aldredge, deputy district director for Rep. Stefani Carter; Rear Adm. Epifiani "Epi" Elizondo, regional health administrator, and Stacy Harper, both with the U.S. Department of Health and Human Services; Pat Thompson, Dallas Cowboys organization; Julie Stefko, youth fitness project manager, NFL Play 60 FITNESSGRAM Project and Kate Losecco, Cooper Institute; Dolores Bell, regional manager, Big Thought; The Texas A&M University System, Dairy MAX and dairy industry representatives; and local dairy farmer and Dairy MAX board member Lynn Ramsey.
More than 60 AgriLife Extension East Region family and consumer sciences agents also participated in event activities, including the 5K and post-activity "refueling" where they were provided bananas, chocolate milk and yogurt for energy.
"Our agency's family and consumer sciences and 4-H and youth development agents already work extensively in Texas schools," said Dr. Susan Ballabina, AgriLife Extension family and consumer sciences regional director for the East Region.
Ballabina helped coordinate the Fuel Up to Play 60 event with Dairy MAX, a nonprofit organization affiliated with the National Dairy Council and funded by dairy farming families in Texas, New Mexico, Oklahoma and southwest Kansas.
"The Fuel Up to Play 60 umbrella will allow us additional visibility and resources to enhance our school-based efforts and increase our visibility," Ballabina said.
She added that health and nutrition is one of the agency's key program areas and AgriLife Extension has an "extensive network of specialists, county agents and volunteers" who work together to serve Texans through community-based education.
Lee, who has visited area schools over the past two years as part of Fuel up to Play 60 program participation, said he was excited about having more people out in the community to spread the word about health and wellness to young people.
"The more people we have out there reaching the kids, the greater the impact of the program and its ability to help them learn the importance of good nutrition and physical exercise," he said.
"The Fuel Up to Play 60 program puts kids in the driver's seat to their own health," said Teresa Wagner, registered dietitian and director of medical outreach for Dairy MAX. "It's our responsibility to create the type of collaborative environment we have in 70,000 schools across the nation, so youth can ultimately apply healthy habits and make nutritious choices throughout their lives. This partnership can help extend our resources and increase our impact."
"We already offer significant learning opportunities to 4-H members interested in health and nutrition," Ballabina said. "The physical activity and nutrition challenges offered through Fuel Up to Play 60 will give our 4-H members more opportunities to learn about health and to positively influence the health of their peers."
Dr. Chris Boleman, Texas 4-H and youth development program director in College Station, who attended the kickoff, said he had already seen the "enthusiasm and excitement" of 4-H members aware of AgriLife Extension's new involvement in the program.
"I know our 4-H members will take the enthusiasm and excitement they're showing about the Fuel Up to Play 60 to others in their clubs and to schools throughout this region and, as it expands, throughout the state," Boleman said.
Boleman noted that currently there are more than 14,000 4-H club members in the agency's East Region and more than 60,000 members statewide. More than 662,000 youth are involved in 4-H programs or activities statewide.
"Our collaborations with other organizations make our network of agents able to accomplish even more, and we couldn't be more pleased to be partnering with Dairy MAX and the NFL to support the Fuel Up to Play 60 program," Ballabina said.


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SCRANTON, PENNSYLVANIA (WKZO) -- Normally this time of the year Derek Jeter is counting up his All-Star votes, but this year he is struggling just to be competitive in the minors, testing that shattered ankle he suffered against the Tigers in the playoffs last season.
Kalamazoo's Yankee says he was actually nervous before his first game back with the Scranton Railriders Saturday.
But he didn't get to try out the ankle much during the game against the Lehigh Valley Iron Pigs. He walked and went 0 for 2 at the plate the other two times he batted. The only ball he fielded was right to him.
He says he is anxious to get to back to New York, but no time table has been set for his return.
He will be on the Railrider's roster when they take to the field Monday evening.


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In a city brimming with practically a film fest a week, the selections might seem overwhelming, but one fest in particular gets our stamp of approval: the 20th annual Environmental Film Festival kicking off today and extending through the end of next week. Among the most well-known films you'll find The Tsunami and the Cherry Blossom, from Academy Award-nominated director Lucy Walker, The Dust Bowl, from iconic filmmaker Ken Burns, and one flick fresh from the 2012 Sundance fest, A Fierce Green Fire: The Battle for a Living Planet.
Tonight alone offers a plethora of flicks to choose from, including the much-anticipated, You've Been Trumped, the self-proclaimed David and Goliath story of our hair idol, Donald Trump, and his showdown with a group of Scottish homeowners determined to bar him from building a gargantuan golf course and luxury hotel on one of Scotland's last protected wilderness areas. Typically, you'd expect the little guys to triumph for the sake of a dramatic ending, but the Scottish government, desperate to bring jobs into the country, is taking Trump's side. Head over to E Street Cinema tonight at 7 p.m. to see whether Scotland will get Trumped.
E Street Cinema, 555 11th Street NW
7:00 p.m.
Tickets: $7.50


text:
Aura Boutique
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Item no.: 220121-0054
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text:
Photographers and media specialists throughout California packed their gear and moved out, but were often thwarted in their attempts to get close to the fire. Certain professionals, however, succeeded.
Striker Captains Marty Greunke and Casey Zenger of West Stanislaus County Fire Department were more concerned with containing the spreading Rim Fire—the 11th largest wildfire in recent California history—than offsetting the camera, but were still nabbed in a couple pictures with a few of their cohorts.
"We were one of the few crews that had our pictures taken because we were working the line," said Greunke, who collaborated with resources from the City of Patterson, Newman and members of Tuolumne County. "We've been in pictures in the L.A. Times and the San Francisco Chronicle."
A photograph by Justin Sullivan shows one unidentified firefighter with the West Stanislaus County Fire Department monitoring the fire from the top of Patterson's OES engine along Highway 120 near Groveland on Aug. 24. The photograph has been added to National Geographic's environment portion, alongside other resplendent shots.
Pictures maybe worth a thousand words, but for Greunke and Zenger, there was only one word to describe the wall of fire—intense.
"It was very destructive," said Zenger, who spent nine days in the fray. "Parts of the forest looked like you were on the moon—just ash."
They also described seeing a plethora of melted signs, skyscraper flames and a charred building. A few of the team members took out their own phones and began to track their journey with pictures and videos as well.
"It was the best experience," said rookie Firefighter Frank Silvia. "It was nice to see how the whole thing works. You learn about it in class, but it's different when you actually see it happen. It makes sense and I learned a lot."
Despite being chronicled in the largest nonprofit scientific and educational institution in the world, the team said their biggest accomplishment was containing the fire from hundreds of threatened homes and families.
Their efforts didn't go unnoticed by the community.
"You should have seen the base," said Greunke. "The firefighters were well taken care of. We had free haircuts given out, and Girl Scouts came by to drop off supplies for us. The level of community support was awesome. People were cheering like we were celebrities all the way from Patterson to the fire."
"But we were just doing our job," added Zenger.
Contact Brooke Borba at 892-6187, ext. 24, or email@example.com.


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I sat on the plane waiting for it to take off and fly the friendly skies. I was a hot, tired, sticky mess and for the first time in two days I wasn't surrounded by people with diabetes. And quite frankly, that just felt weird and lonely all rolled into one.
I was on my way home from the Roche Summit and I was sorry to leave Indy for so many reasons.
As I sat there lost in thought and Fruit Zombies, my pump chirped out a familiar array of beeps reminding me that I had indeed had a temporary basal rate going on. I glanced down at it and saw that it was slightly entangled in my seatbelt, fixed it and tried to concentrate on the Fruit Zombies on my iPad.
Two minutes later I heard the familiar chirping again and got really annoyed.
In my head I was like: COME ON INSULIN PUMP- I know that you're running a temporary basal rate - I'm the one who programmed the temporary basal rate - SHUT UP ALREADY. Except this time when I glanced down at my pump to see what it was squawking about, I realized my pump wasn't the one chirping.
In my head I was like: COME ON INSULIN PUMP- I know that you're running a temporary basal rate - I'm the one who programmed the temporary basal rate - SHUT UP ALREADY. Except this time when I glanced down at my pump to see what it was squawking about, I realized my pump wasn't the one chirping.
And I was totally stunned.
HOLY CRAP, I thought - Someone else on this plane has diabetes. I Iooked around and saw a woman in the aisle one row behind and across looking at her pump and quickly tucking it back into her shirt, lean back and close her eyes.
I didn't want to interrupt her if she was trying to sleep, but I was dying to jump across the isle and say: Hey, I have insulin pump too, I have an insulin pump, too!!!!
But I didn't want to risk waking her. Seriously, if she was as tired as I was - she was probably already asleep.
The flight attendant came around and told me to put my iPad away and before I knew it the plane was took off. Once we reached "cruising altitude" I glanced over at Insulin Pump Girl again and she was fast asleep.
And I was bummed out. But I was also really tired and couldn't keep my eyes open .
Then I closed my eyes and fell promptly asleep for about an hour and fifteen minutes - And I never sleep on planes so that's saying something!
I woke up once, when my pump reminded me that I less than 20 units left and was in and out of sleep fully after that. When I finally woke up, we were probably a half hour from landing, but just to be sure I took out my pump and glanced at the clock, then I glanced over towards Insulin Pump Girl because I thought I felt someone watching me - And she was.
Insulin Pump Girl: You have a pump too!!! I heard beeping earlier and was trying to catch your eye!
Me: Oh my God, I heard your pump beeping earlier and tried to catch your eye too! But you were trying to sleep and I didn't want to bother you....
Insulin Pump Girl: That's so funny!
Me: RIGHT! This is so cool!
Insulin Pump Girl: Were you in town for AADE?
Me: No, I had was at the Roche Summit - But I wish I could have stayed for AADE.
Insulin Pump Girl: I was in town for some AADE meetings -I'm a Diabetes Pediatric Nurse.
Me; YOU'RE AWESOME.
And then we talked about how long we'd had diabetes (she was 27 years in ) and how the diabetes world has changed and how amazing "diabetes in the wild" is!
Her seat mate (an older lady with a terrific smile) just kept watching our exchange, grinned from ear to ear and didn't say a word.
Diabetes Pump Girl knew about the Diabetes On-Line Community and and we chatted away until it was time to land.
We walked through the terminal together and continued talking. Finally we exchanged cards, hugged one another, said our goodbyes and went our separate ways. She had to find her connecting flight and I went on to find my luggage.
And as waited for the carousel to deliver my big purple suitcase I couldn't help but smile and be happy about what had just happened.
Diabetes in the wild, diabetics on a plane, D-Meet in the sky - Whatever you want to call it, it was a wonderful and awesome thing!


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HEAT oven to 375°F.
PLACE squash halves in microwaveable dish; cover with vented microwaveable plastic wrap. Microwave on HIGH 12 to 14 min. or until tender, turning after 6 min. Let stand, covered, 5 min.
SCOOP squash from shells, leaving 1/4-inch-thick shells. Mash squash with margarine; spoon into shells. Place on baking sheet; top each squash half with 1 tsp. brown sugar and 1/4 cup marshmallows.
BAKE 12 to 15 min. or until squash is heated through and marshmallows are lightly browned.


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3461 Posts in 1065 Topics by 740 members
Data Model Questions
SilverStripe Forums » Data Model Questions » Populate Data directly
Moderators: martimiz, Sean, biapar, Willr, Ingo, swaiba, simon_w
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Populate Data directly
17 March 2011 at 7:50am
Hoping someone can help
I am using ModelAdmin to manage custom DataObjects. I have used a script to directly populate the tables (add additional) with necessary data ( as I have large volume of data to be fed into the site ).
All seems ok when I review the tables, however when I use the ModelAdmin interface to list them it only lists the original items ( added via cms interface ) and not what I have added direclty.
Community Member
2 Posts
Re: Populate Data directly
17 March 2011 at 10:45am
Have you run the dev/build (as in, go to the url yoursite/dev/build)? After you make changes to the underlying code, dev/build will update DB tables.
Going the other way (from new date to updating views), be sure to flush cache (e.g yoursite/?flush=1)
Also, if you have uploaded any files directly, you will need to go to the Files and Images section of the CMS and click the "Look for new files" button to let SS know about your new files.
Re: Populate Data directly
17 March 2011 at 11:47am Last edited: 17 March 2011 8:00pm
thanks for your feedback. yes I have cleared the cache using flush, done the rebuild and also cleared the silverstripe cache folder completely.
Whats interesting is that if I add an item via the CMS interface after data import via script, then Add button generates an errors. However when I revisit the full list, it shows an entry from what I had imported via script.
To clarify that a bit furtehr, if I had 5 items before script import and the import added 3 items, then it will only show the original 5 items. When I add the 6th item via cms, it shows the first item from the imported data insstead...
A thought:
Is it possible, SS might be keeping the last used incremental value somewhere which is updated when adding via CMS?
Re: Populate Data directly
17 March 2011 at 10:46pm Last edited: 17 March 2011 10:49pm
I have solved my problem. The issue was with the way my unique ID was being created. To elaborate a bit more for anyone else attempting to do the same.
My goal was to pull data from an existing SS site for specific DataObjects only. These DO also had has_many relations. Since the db I was migrating to already contains data ( and potentially duplicate ids if attempted to migrate as is ), I had to generate the Ids myself.
Both my main Object and its has_many objects is extended of a custom DO to use common methods. What was key for me is in understanding that SS kept uniqu id at the custom DO level.
I chose to use custom script as the schema was the same and my attempt at exploring the use of CsvBulkLoader with has_many came up futile.
Community Member
2 Posts
Re: Populate Data directly
18 March 2011 at 2:36am
Thanks for posting your solution!
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Nastiuk, 23, began the season with Gwinnett, going 4-2-2 with a 3.04 goals-against-average and a .886 save percentage over eight games since being released from Providence's training camp on October 7.
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Providence Bruins season tickets, Flex Tix packages and single game tickets are on sale now for the 2008-09 season. To purchase tickets or to learn more information, call the P-Bruins ticket office at (401) 273-5000 or log on to the team website at providencebruins.com.


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