LAVA: An Open-Source Approach To Designing LAMP (Loop-Mediated Isothermal Amplification) DNA Signatures

<p>Abstract</p> <p>Background</p> <p>We developed an extendable open-source Loop-mediated isothermal AMPlification (LAMP) signature design program called LAVA (LAMP Assay Versatile Analysis). LAVA was created in response to limitations of existing LAMP signature programs.</p> <p>Results</p> <p>LAVA identifies combinations of six primer regions for basic LAMP signatures, or combinations of eight primer regions for LAMP signatures with loop primers, which can be used as LAMP signatures. The identified primers are conserved among target organism sequences. Primer combinations are optimized based on lengths, melting temperatures, and spacing among primer sites. We compare LAMP signature candidates for <it>Staphylococcus aureus </it>created both by LAVA and by PrimerExplorer. We also include signatures from a sample run targeting all strains of <it>Mycobacterium tuberculosis</it>.</p> <p>Conclusions</p> <p>We have designed and demonstrated new software for identifying signature candidates appropriate for LAMP assays. The software is available for download at <url>http://lava-dna.googlecode.com/</url>.</p

New Insights into the Genetic Regulation of Homologue Disjunction in Mammalian Oocytes

Mammalian oocytes execute a unique meiotic programme involving 2 arrest stages and an unusually protracted preamble to chromosome segregation during the first meiotic division (meiosis I). How mammalian oocytes successfully navigate their exceptional meiotic journey has long been a question of immense interest. Understanding the minutiae of female mammalian meiosis I is not merely of academic interest as 80–90% of human aneuploidy is the consequence of errors arising at this particular stage of oocyte maturation, a stage with a peculiar vulnerability to aging. Recent evidence indicates that oocytes employ many of the same cast of proteins during meiosis I as somatic cells do during mitosis, often to execute similar tasks, but intriguingly, occasionally delegate them to unexpected and unprecedented roles. This is epitomised by the master cell-cycle regulon, the anaphase-promoting complex or cyclosome (APC/C), acting in concert with a critical APC/C-targeted surveillance mechanism, the spindle assembly checkpoint (SAC). Together, the APC/C and the SAC are among the most influential entities overseeing the fidelity of cell-cycle progression and the precision of chromosome segregation. Here I review the current status of pivotal elements underpinning homologue disjunction in mammalian oocytes including spindle assembly, critical biochemical anaphase-initiating events, APC/C activity and SAC signalling along with contemporary findings relevant to progressive oocyte SAC dysfunction as a model for age-related human aneuploidy

Reelin Is Involved in Transforming Growth Factor-β1-Induced Cell Migration in Esophageal Carcinoma Cells

Reelin (RELN), which is a glycoprotein secreted by Cajal-Retzius cells of the developing cerebral cortex, plays an important role in neuronal migration, but its role in cell migration and cancer metastasis is largely unclear. Here, we showed that cell motility was significantly increased in KYSE-510 cells by TGF-β1 treatment. Moreover, TGF-β1 decreased RELN mRNA expression and overexpression of Reelin at least partly reversed TGF-β1-induced cell migration in KYSE-30 cells. Furthermore, this negative regulation of Reelin expression by TGF-β1 was through Snail, one transcription factor which was induced by TGF-β1 in KYSE-510 cells. RELN promoter activity was reduced in parallel with the induction of Snail after TGF-β1 treatment and Snail suppressed both RELN promoter activity and expression through binding to E-box sequences in the RELN promoter region in ESCC cells. Knockdown of RELN induced cell migration in KYSE-510 cells, together with the increase of mesenchymal markers expression. Taken together, Reelin is an essential negative regulator in the TGF-β1-induced cell migration process, and is suppressed by TGF-β pathway at the transcriptional level through Snail regulation. Therefore, the correlation of Reelin and TGF-β pathway was critical in cancer metastasis, and Reelin could be one potential anti-metastasis target in future clinical practice

Cdc20 Is Critical for Meiosis I and Fertility of Female Mice

Chromosome missegregation in germ cells is an important cause of unexplained infertility, miscarriages, and congenital birth defects in humans. However, the molecular defects that lead to production of aneuploid gametes are largely unknown. Cdc20, the activating subunit of the anaphase-promoting complex/cyclosome (APC/C), initiates sister-chromatid separation by ordering the destruction of two key anaphase inhibitors, cyclin B1 and securin, at the transition from metaphase to anaphase. The physiological significance and full repertoire of functions of mammalian Cdc20 are unclear at present, mainly because of the essential nature of this protein in cell cycle progression. To bypass this problem we generated hypomorphic mice that express low amounts of Cdc20. These mice are healthy and have a normal lifespan, but females produce either no or very few offspring, despite normal folliculogenesis and fertilization rates. When mated with wild-type males, hypomorphic females yield nearly normal numbers of fertilized eggs, but as these embryos develop, they become malformed and rarely reach the blastocyst stage. In exploring the underlying mechanism, we uncover that the vast majority of these embryos have abnormal chromosome numbers, primarily due to chromosome lagging and chromosome misalignment during meiosis I in the oocyte. Furthermore, cyclin B1, cyclin A2, and securin are inefficiently degraded in metaphase I; and anaphase I onset is markedly delayed. These results demonstrate that the physiologically effective threshold level of Cdc20 is high for female meiosis I and identify Cdc20 hypomorphism as a mechanism for chromosome missegregation and formation of aneuploid gametes

Separase Phosphosite Mutation Leads to Genome Instability and Primordial Germ Cell Depletion during Oogenesis

To ensure equal chromosome segregation and the stability of the genome during cell division, Separase is strictly regulated primarily by Securin binding and inhibitory phosphorylation. By generating a mouse model that contained a mutation to the inhibitory phosphosite of Separase, we demonstrated that mice of both sexes are infertile. We showed that Separase deregulation leads to chromosome mis-segregation, genome instability, and eventually apoptosis of primordial germ cells (PGCs) during embryonic oogenesis. Although the PGCs of mutant male mice were completely depleted, a population of PGCs from mutant females survived Separase deregulation. The surviving PGCs completed oogenesis but produced deficient initial follicles. These results indicate a sexual dimorphism effect on PGCs from Separase deregulation, which may be correlated with a gender-specific discrepancy of Securin. Our results reveal that Separase phospho-regulation is critical for genome stability in oogenesis. Furthermore, we provided the first evidence of a pre-zygotic mitotic chromosome segregation error resulting from Separase deregulation, whose sex-specific differences may be a reason for the sexual dimorphism of aneuploidy in gametogenesis

A-002 (Varespladib), a phospholipase A2 inhibitor, reduces atherosclerosis in guinea pigs

<p>Abstract</p> <p>Background</p> <p>The association of elevated serum levels of secretory phospholipase A₂(sPLA₂) in patients with cardiovascular disease and their presence in atherosclerotic lesions suggest the participation of sPLA₂enzymes in this disease. The presence of more advanced atherosclerotic lesions in mice that overexpress sPLA₂enzymes suggest their involvement in the atherosclerotic process. Therefore, the sPLA₂family of enzymes could provide reasonable targets for the prevention and treatment of atherosclerosis. Thus, A-002 (varespladib), an inhibitor of sPLA₂enzymes, is proposed to modulate the development of atherosclerosis.</p> <p>Methods</p> <p>Twenty-four guinea pigs were fed a high saturated fat, high cholesterol diet (0.25%) for twelve weeks. Animals were treated daily with A-002 (n = 12) or vehicle (10% aqueous acacia; n = 12) by oral gavage. After twelve weeks, animals were sacrificed and plasma, heart and aorta were collected. Plasma lipids were measured by enzymatic methods, lipoprotein particles size by nuclear magnetic resonance, aortic cytokines by a colorimetric method, and aortic sinus by histological analyses.</p> <p>Results</p> <p>Plasma total cholesterol, HDL cholesterol and triglycerides were not different among groups. However, the levels of inflammatory cytokines interleukin (IL)-10, IL-12 and granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly reduced in the treatment group. This group also had a significant 27% reduction in cholesterol accumulation in aorta compared with placebo group. Morphological analysis of aortic sinus revealed that the group treated with A-002 reduced atherosclerotic lesions by 24%.</p> <p>Conclusion</p> <p>The use of A-002 may have a beneficial effect in preventing diet-induced atherosclerosis in guinea pigs.</p

Identification of a Classical Bipartite Nuclear Localization Signal in the Drosophila TEA/ATTS Protein Scalloped

Drosophila melanogaster wing development has been shown to rely on the activity of a complex of two proteins, Scalloped (Sd) and Vestigial (Vg). Within this complex, Sd is known to provide DNA binding though its TEA/ATTS domain, while Vg modulates this binding and provides transcriptional activation through N- and C-terminal activation domains. There is also evidence that Sd is required for the nuclear translocation of Vg. Indeed, a candidate sequence which shows consensus to the bipartite family of nuclear localization signals (NLSs) has been identified within Sd previously, though it is not known if it is functional, or if additional unpredicted signals that mediate nuclear transport exist within the protein. By expressing various enhanced green fluorescent protein (eGFP) tagged constructs within Drosophila S2 cells, we demonstrate that this NLS is indeed functional and necessary for the proper nuclear localization of Sd. Additionally, the region containing the NLS is critical for the wildtype function of ectopically expressed Sd, in the context of wing development. Using site-directed mutagenesis, we have identified a group of five amino acids within this NLS which is critical for its function, as well as another group of two which is of lesser importance. Together with data that suggests that this sequence mediates interactions with Importin-α3, we conclude that the identified NLS is likely a classical bipartite signal. Further dissection of Sd has also revealed that a large portion of the C-terminal domain of the protein is required its proper nuclear localization. Finally, a Leptomycin B (LB) sensitive signal which appears to facilitate nuclear export is identified, raising the possibility that Sd also contains a nuclear export signal (NES)