Permanent genetic resources added to Molecular Ecology Resources Database 1 October 2010-30 November 2010

This article documents the addition of 277 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Ascochyta rabiei, Cambarellus chapalanus, Chionodraco hamatus, Coptis omeiensis, Cynoscion nebulosus, Daphnia magna, Gerbillus nigeriae, Isurus oxyrinchus, Lates calcarifer, Metacarcinus magister, Oplegnathus fasciatus, Pachycondyla verenae, Phaethon lepturus, Pimelodus grosskopfii, Rotylenchulus reniformis, Scomberomorus niphonius, Sepia esculenta, Terapon jarbua, Teratosphaeria cryptica and Thunnus obesus. These loci were cross-tested on the following species: Austropotamobius italicus, Cambarellus montezumae, Cambarellus puer, Cambarellus shufeldtii, Cambarellus texanus, Chionodraco myersi, Chionodraco rastrospinosus, Coptis chinensis, Coptis chinensis var. brevisepala, Coptis deltoidea, Coptis teeta, Orconectes virilis, Pacifastacus leniusculus, Pimelodus bochii, Procambarus clarkii, Pseudopimelodus bufonius, Rhamdia quelen, Sepia andreana, Sepiella maindroni, Thunnus alalunga, Thunnus albacares, Thunnus maccoyii, Thunnus orientalis, Thunnus thynnus and Thunnus tonggol.     

Ectopic pregnancy as a model to identify endometrial genes and signaling pathways important in decidualization and regulated by local trophoblast

The endometrium in early pregnancy undergoes decidualization and functional changes induced by local trophoblast, which are not fully understood. We hypothesized that endometrium from tubal ectopic pregnancy (EP) could be interrogated to identify novel genes and pathways involved in these processes. Gestation-matched endometrium was collected from women with EP (n = 11) and intrauterine pregnancies (IUP) (n = 13). RNA was extracted from the tissue. In addition, tissues were prepared for histological analysis for degree of decidualization. We compared a) the samples from EP that were decidualized (n = 6) with non-decidualized samples (n = 5), and b) the decidualized EP (n = 6) with decidualization-matched IUP (n = 6) samples using an Affymetrix gene array platform, with Ingenuity Pathway Analysis, combined with quantitative RT-PCR. Expression of PRL and IGFBP1 was used to confirm the degree of decidualization in each group. There were no differences in PRL or IGFBP1 expression in the decidualization-matched samples but a marked reduction (P<0.001) in the non-decidualized samples. Decidualization was associated with increased expression of 428 genes including SCARA5 (181-fold), DKK1 (71-fold) and PROK1 (32-fold), and decreased expression of 230 genes including MMP-7 (35-fold) and SFRP4 (21-fold). The top canonical pathways associated with these differentially expressed genes were Natural Killer Cell and Wnt/b-Catenin signaling. Local trophoblast was associated with much less alteration of endometrial gene expression with an increase in 56 genes, including CSH1 (8-fold), and a reduction in 29 genes including CRISP3 (8-fold). The top associated canonical pathway was Antigen Presentation. The study of endometrium from tubal EP may promote novel insights into genes involved in decidualization and those influenced by factors from neighboring trophoblast. This has afforded unique information not highlighted by previous studies and adds to our understanding of the endometrium in early pregnancy.     

Targeted delivery of neural stem cells to the brain using MRI-guided focused ultrasound to disrupt the blood-brain barrier

Stem cell therapy is a promising strategy to treat neurodegenerative diseases, traumatic brain injury, and stroke. For stem cells to progress towards clinical use, the risks associated with invasive intracranial surgery used to deliver the cells to the brain, needs to be reduced. Here, we show that MRI-guided focused ultrasound (MRIgFUS) is a novel method for non-invasive delivery of stem cells from the blood to the brain by opening the blood brain barrier (BBB) in specific brain regions. We used MRI guidance to target the ultrasound beam thereby delivering the iron-labeled, green fluorescent protein (GFP)-expressing neural stem cells specifically to the striatum and the hippocampus of the rat brain. Detection of cellular iron using MRI established that the cells crossed the BBB to enter the brain. After sacrifice, 24 hours later, immunohistochemical analysis confirmed the presence of GFP-positive cells in the targeted brain regions. We determined that the neural stem cells expressed common stem cell markers (nestin and polysialic acid) suggesting they survived after transplantation with MRIgFUS. Furthermore, delivered stem cells expressed doublecortin in vivo indicating the stem cells were capable of differentiating into neurons. Together, we demonstrate that transient opening of the BBB with MRIgFUS is sufficient for transplantation of stem cells from the blood to targeted brain structures. These results suggest that MRIgFUS may be an effective alternative to invasive intracranial surgery for stem cell transplantation.     

Crosstalk between EET and HO-1 downregulates Bach1 and adipogenic marker expression in mesenchymal stem cell derived adipocytes

Epoxygenase activity and synthesis of epoxyeicosatrienoic acids (EETs) have emerged as important modulators of obesity and diabetes. We examined the effect of the EET-agonist 12-(3-hexylureido)dodec-8(2) enoic acid on mesenchymal stem cell (MSC) derived adipocytes proliferation and differentiation. MSCs expressed substantial levels of EETs and inhibition of soluble epoxide hydrolase (sEH) increased the level of EETs and decreased adipogenesis. EET agonist treatment increased HO-1 expression by inhibiting a negative regulator of HO-1 expression, Bach-1. EET treatment also increased βcatenin and pACC levels while decreasing PPARγ C/EBPα and fatty acid synthase levels. These changes were manifested by a decrease in the number of large inflammatory adipocytes, TNFα, IFNγ and IL-1α, but an increase in small adipocytes and in adiponectin levels. In summary, EET agonist treatment inhibits adipogenesis and decreases the levels of inflammatory cytokines suggesting the potential action of EETs as intracellular lipid signaling modulators of adipogenesis and adiponectin.     

High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing

RNA interference (RNAi) screening is a state-of-the-art technology that enables the dissection of biological processes and disease-related phenotypes. The commercial availability of genome-wide, short hairpin RNA (shRNA) libraries has fueled interest in this area but the generation and analysis of these complex data remain a challenge. Here, we describe complete experimental protocols and novel open source computational methodologies, shALIGN and shRNAseq, that allow RNAi screens to be rapidly deconvoluted using next generation sequencing. Our computational pipeline offers efficient screen analysis and the flexibility and scalability to quickly incorporate future developments in shRNA library technology.     

Funding begets biodiversity

Aim Effective conservation of biodiversity relies on an unbiased knowledge of its distribution. Conservation priority assessments are typically based on the levels of species richness, endemism and threat. Areas identified as important receive the majority of conservation investments, often facilitating further research that results in more species discoveries. Here, we test whether there is circularity between funding and perceived biodiversity, which may reinforce the conservation status of areas already perceived to be important while other areas with less initial funding may remain overlooked. Location Eastern Arc Mountains, Tanzania. Methods We analysed time series data (1980–2007) of funding (n = 134 projects) and plant species records (n = 75,631) from a newly compiled database. Perceived plant diversity, over three decades, is regressed against funding and environmental factors, and variances decomposed in partial regressions. Cross‐correlations are used to assess whether perceived biodiversity drives funding or vice versa. Results Funding explained 65% of variation in perceived biodiversity patterns – six times more variation than accounted for by 34 candidate environmental factors. Cross‐correlation analysis showed that funding is likely to be driving conservation priorities and not vice versa. It was also apparent that investment itself may trigger further investments as a result of reduced start‐up costs for new projects in areas where infrastructure already exists. It is therefore difficult to establish whether funding, perceived biodiversity, or both drive further funding. However, in all cases, the results suggest that regional assessments of biodiversity conservation importance may be biased by investment. Funding effects might also confound studies on mechanisms of species richness patterns. Main conclusions Continued biodiversity loss commands urgent conservation action even if our knowledge of its whereabouts is incomplete; however, by concentrating inventory funds in areas already perceived as important in terms of biodiversity and/or where start‐up costs are lower, we risk losing other areas of underestimated or unknown value.     

AP-1 and clathrin are essential for secretory granule biogenesis in Drosophila

Regulated secretion of hormones, digestive enzymes, and other biologically active molecules requires the formation of secretory granules. Clathrin and the clathrin adaptor protein complex 1 (AP-1) are necessary for maturation of exocrine, endocrine, and neuroendocrine secretory granules. However, the initial steps of secretory granule biogenesis are only minimally understood. Powerful genetic approaches available in the fruit fly Drosophila melanogaster were used to investigate the molecular pathway for biogenesis of the mucin-containing "glue granules" that form within epithelial cells of the third-instar larval salivary gland. Clathrin and AP-1 colocalize at the trans-Golgi network (TGN) and clathrin recruitment requires AP-1. Furthermore, clathrin and AP-1 colocalize with secretory cargo at the TGN and on immature granules. Finally, loss of clathrin or AP-1 leads to a profound block in secretory granule formation. These findings establish a novel role for AP-1- and clathrin-dependent trafficking in the biogenesis of mucin-containing secretory granules.     

Self-catalyzed degradable cationic polymer for release of DNA

The controlled release of siRNA or DNA complexes from cationic polymers is an important parameter design in polymer-based delivery carriers. In this work, we use the self-catalyzed degradable poly(2-dimethylaminoethyl acrylate) (PDMAEA) to strongly bind, protect, and then release oligo DNA (a mimic for siRNA) without the need for a cellular or external trigger. This self-catalyzed hydrolysis process of PDMAEA forms poly(acrylic acid) and N,N'-dimethylamino ethyl ethanol, both of which have little or no toxicity to cells, and offers the advantage of little or no toxicity to off-target cells and tissues. We found that PDMAEA makes an ideal component of a delivery carrier by protecting the oligo DNA for a sufficiently long period of time to transfect most cells (80% transfection after 4 h) and then has the capacity to release the DNA inside the cells after ~10 h. The PDMAEA formed large nanoparticle complexes with oligo DNA of ~400 nm that protected the oligo DNA from DNase in serum. The nanoparticle complexes showed no toxicity for all molecular weights at a nitrogen/phosphorus (N/P) ratio of 10. Only the higher molecular weight polymers at very high N/P ratios of 200 showed significant levels of cytotoxicity. These attributes make PDMAEA a promising candidate as a component in the design of a gene delivery carrier without the concern about accumulated toxicity of nanoparticles in the human body after multiadministration, an issue that has become increasingly more important.