Addgene: Making Materials Sharing “Science As Usual”

Addgene (www.addgene.org) is a nonprofit organization that facilitates biomedical research and discovery by improving access to useful research materials and information. To fulfill this mission, Addgene works with hundreds of laboratories all over the world to collect high-quality published plasmids and data for the repository that can then be distributed to academic institutions and used to further research. Biological resource centers such as Addgene are an important part of the scientific infrastructure. They play a key role in helping scientists overcome logistical barriers to sharing, improving experimental reproducibility, and optimizing use of limited resources.     

Modified dsRNAs that are not processed by Dicer maintain potency and are incorporated into the RISC

Chemical modification of RNA duplexes can provide practical advantages for RNA interference (RNAi) triggering molecules including increased stability, safety and specificity. The impact of nucleotide modifications on Dicer processing, RISC loading and RNAi-mediated mRNA cleavage was investigated with duplexes ≥25 bp in length. It is known that dsRNAs ≥25 bp are processed by Dicer to create classic 19-bp siRNAs with 3′-end overhangs. We demonstrate that the presence of minimal modification configurations on longer RNA duplexes can block Dicer processing and result in the loading of the full-length guide strand into RISC with resultant mRNA cleavage at a defined site. These longer, modified duplexes can be highly potent gene silencers, with EC50s in the picomolar concentration range, demonstrating that Dicer processing is not required for incorporation into RISC or potent target silencing.     

Relocation of a Ca2+-dependent protein kinase activity during pollen tube reorientation

Pollen tube reorientation is a dynamic cellular event that is crucial for successful fertilization. We have shown previously that pollen tube orientation is regulated by cytosolic free calcium ([Ca2+]c). In this paper, we studied the activity of a Ca2+-dependent protein kinase during reorientation. The kinase activity was assayed in living cells by using confocal ratio imaging of BODIPY FL bisindolylmaleimide. We found that growing pollen tubes exhibited higher protein kinase activity in the apical region, whereas nongrowing cells showed uniform distribution. Modification of growth direction by diffusion of inhibitors/activators from a micropipette showed the spatial redistribution of kinase activity to predict the new growth orientation. Localized increases in [Ca2+]c induced by photolysis of caged Ca2+ that led to reorientation also increased kinase activity. Molecular and immunological assays suggest that this kinase may show some functional homology with protein kinase C. We suggest that the tip-localized gradient of kinase activity promotes Ca2+-mediated exocytosis and may act to regulate Ca2+ channel activity.     

Cancer therapy

In recent years a growing recognition that molecularly-targeted therapies face formidable obstacles has revived interest in more generic tumor cell phenotypes that could be exploited for therapy. Two recent reports demonstrate that cancer cell survival is critically dependent on the activity of MTH1, a nucleotide pyrophosphatase that converts the oxidized nucleotides 8-oxo-dGTP and 2-OH-dATP to the corresponding monophosphates, thus preventing their incorporation into genomic DNA. Tumor cells frequently overexpress MTH1, probably because malignant transformation creates oxidative stress that renders the nucleotide pool highly vulnerable to oxidation. As a result, MTH1 inhibition in cancer cells results in accumulation and incorporation of 8-oxo-dGTP and 2-OH-dATP into DNA, leading to DNA damage and cell death. This toxic effect is highly cancer cell-specific, as MTH1 is generally dispensable for the survival of normal, untransformed cells. Importantly, MTH1 proves to be a “druggable” enzyme that can be inhibited both by an existing protein kinase inhibitor drug, crizotinib, and by novel compounds identified through screening. Inhibition of MTH1 leading to toxic accumulation of oxidized nucleotides specifically in tumor cells therefore represents an example of a “non-personalised” approach to cancer therapy.     

Nominal association with CHRNA4 variants and nicotine dependence

Nicotine binds to nicotinic acetylcholine receptors and studies in animal models have shown that α4β2 receptors mediate many behavioral effects of nicotine. Human genetics studies have provided support that variation in the gene that codes for the α4 subunit influences nicotine dependence (ND), but the evidence for the involvement of the β2 subunit gene is less convincing. In this study, we examined the genetic association between variation in the genes that code for the α4 (CHRNA4) and β2 (CHRNB2) subunits of the nicotinic acetylcholine receptor and a quantitative measure of lifetime DSM‐IV ND symptom counts. We performed this analysis in two longitudinal family‐based studies focused on adolescent antisocial drug abuse: the Center on Antisocial Drug Dependence (CADD, N = 313 families) and Genetics of Antisocial Drug Dependence (GADD, N = 111 families). Family‐based association tests were used to examine associations between 14 single nucleotide polymorphisms (SNPs) in CHRNA4 and CHRNB2 and ND symptoms. Symptom counts were corrected for age, sex and clinical status prior to the association analysis. Results, when the samples were combined, provided modest evidence that SNPs in CHRNA4 are associated with ND. The minor allele at both rs1044394 (A; Z = 1.988, P = 0.047, unadjusted P‐value) and rs1044396 (G; Z = 2.398, P = 0.017, unadjusted P‐value) was associated with increased risk of ND symptoms. These data provide suggestive evidence that variation in the α4 subunit of the nicotinic acetylcholine receptor may influence ND liability.     

Genome size evolution of the extant lycophytes and ferns

Ferns and lycophytes have remarkably large genomes. However, little is known about how their genome size evolved in fern lineages. To explore the origins and evolution of chromosome numbers and genome size in ferns, we used flow cytometry to measure the genomes of 240 species (255 samples) of extant ferns and lycophytes comprising 27 families and 72 genera, of which 228 species (242 samples) represent new reports. We analyzed correlations among genome size, spore size, chromosomal features, phylogeny, and habitat type preference within a phylogenetic framework. We also applied ANOVA and multinomial logistic regression analysis to preference of habitat type and genome size. Using the phylogeny, we conducted ancestral character reconstruction for habitat types and tested whether genome size changes simultaneously with shifts in habitat preference. We found that 2C values had weak phylogenetic signal, whereas the base number of chromosomes (x) had a strong phylogenetic signal. Furthermore, our analyses revealed a positive correlation between genome size and chromosome traits, indicating that the base number of chromosomes (x), chromosome size, and polyploidization may be primary contributors to genome expansion in ferns and lycophytes. Genome sizes in different habitat types varied significantly and were significantly correlated with habitat types; specifically, multinomial logistic regression indicated that species with larger 2C values were more likely to be epiphytes. Terrestrial habitat is inferred to be ancestral for both extant ferns and lycophytes, whereas transitions to other habitat types occurred as the major clades emerged. Shifts in habitat types appear be followed by periods of genomic stability. Based on these results, we inferred that habitat type changes and multiple whole-genome duplications have contributed to the formation of large genomes of ferns and their allies during their evolutionary history.     

尊重斯德哥尔摩的历史中心—骑士岛的激活改造

瑞典国家财产委员会拥有骑士岛的所有权与管理权，并计划对该岛上所有的公共空间进行更新和开发，以提高其可达性与吸引力。该项目的核心是找到一种更新和修复岛屿的方法，从而在尊重历史价值的同时满足现代功能需求。对骑士岛南部的改造是岛上公共空间更新的第一部分。设计的关键条件是沿滨水区域创造可以供人步行与停坐的大面积空间，并在保持开放海港氛围的同时，对旧的道路铺装进行管理。设计者设计了一套灵活使用公共空间的综合解决方案，将开放空间与之前的码头一样，沿着水滨的形态进行布局。