A Human Islet Cell-Culture System for High-Throuput screening.

A small-molecule inducer of beta-cell proliferation in human islets represents a potential regeneration strategy for treating type 1 diabetes. However, the lack of suitable human beta cell lines makes such a discovery a challenge. Here, we adapted an islet cell culture system to high-throughput screening to identify such small molecules. We prepared microtiter plates containing extracellular matrix from a human bladder carcinoma cell line. Dissociated human islets were seeded onto these plates, cultured for up to 7 days, and assessed for proliferation by simultaneous Ki67 and C-peptide immunofluorescence. Importantly, this environment preserved beta-cell physiological function, as measured by glucose-stimulated insulin secretion. Adenoviral overexpression of cdk-6 and cyclin D(1), known inducers of human beta cell proliferation, was used as a positive control in our assay. This induction was inhibited by cotreatment with rapamycin, an immunosuppressant often used in islet transplantation. We then performed a pilot screen of 1280 compounds, observing some phenotypic effects on cells. This high-throughput human islet cell culture method can be used to assess various aspects of beta-cell biology on a relatively large number of compounds

Mechanistic Study Of The Photochemical Hydroxide Ion Release From 9-hydroxy-10-methyl-9-phenyl-9,10-dihydroacridine

The excited-state behavior of 9-hydroxy-10-methyl-9-phenyl-9,10-dihydroacridine and its derivative, 9-methoxy-10-methyl-9-phenyl-9,10-dihydroacridine (AcrOR, R = H, Me), was studied via femtosecond and nanosecond UV-vis transient absorption spectroscopy. The solvent effects on C-O bond cleavage were clearly identified: a fast heterolytic cleavage (tau = 108 ps) was observed in protic solvents, while intersystem crossing was observed in aprotic solvents. Fast heterolysis generates 10methyl-9-phenylacridinium (Acr(+)) and -OH, which have a long recombination lifetime (no signal decay was observed within 100 mu s). AcrOH exhibits the characteristic behavior needed for its utilization as a chromophore in the pOH jump experiment

Community annotation and bioinformatics workforce development in concert—Little Skate Genome Annotation Workshops and Jamborees

Recent advances in high-throughput DNA sequencing technologies have equipped biologists with a powerful new set of tools for advancing research goals. The resulting flood of sequence data has made it critically important to train the next generation of scientists to handle the inherent bioinformatic challenges. The North East Bioinformatics Collaborative (NEBC) is undertaking the genome sequencing and annotation of the little skate (Leucoraja erinacea) to promote advancement of bioinformatics infrastructure in our region, with an emphasis on practical education to create a critical mass of informatically savvy life scientists. In support of the Little Skate Genome Project, the NEBC members have developed several annotation workshops and jamborees to provide training in genome sequencing, annotation and analysis. Acting as a nexus for both curation activities and dissemination of project data, a project web portal, SkateBase (http://skatebase.org) has been developed. As a case study to illustrate effective coupling of community annotation with workforce development, we report the results of the Mitochondrial Genome Annotation Jamborees organized to annotate the first completely assembled element of the Little Skate Genome Project, as a culminating experience for participants from our three prior annotation workshops. We are applying the physical/virtual infrastructure and lessons learned from these activities to enhance and streamline the genome annotation workflow, as we look toward our continuing efforts for larger-scale functional and structural community annotation of the L. erinacea genome

Phenothiourea Sensitizes Zebrafish Cranial Neural Crest and Extraocular Muscle Development to Changes in Retinoic Acid and IGF Signaling

1-phenyl 2-thiourea (PTU) is a tyrosinase inhibitor commonly used to block pigmentation and aid visualization of zebrafish development. At the standard concentration of 0.003% (200 µM), PTU inhibits melanogenesis and reportedly has minimal other effects on zebrafish embryogenesis. We found that 0.003% PTU altered retinoic acid and insulin-like growth factor (IGF) regulation of neural crest and mesodermal components of craniofacial development. Reduction of retinoic acid synthesis by the pan-aldehyde dehydrogenase inhibitor diethylbenzaldehyde, only when combined with 0.003% PTU, resulted in extraocular muscle disorganization. PTU also decreased retinoic acid-induced teratogenic effects on pharyngeal arch and jaw cartilage despite morphologically normal appearing PTU-treated controls. Furthermore, 0.003% PTU in combination with inhibition of IGF signaling through either morpholino knockdown or pharmacologic inhibition of tyrosine kinase receptor phosphorylation, disrupted jaw development and extraocular muscle organization. PTU in and of itself inhibited neural crest development at higher concentrations (0.03%) and had the greatest inhibitory effect when added prior to 22 hours post fertilization (hpf). Addition of 0.003% PTU between 4 and 20 hpf decreased thyroxine (T4) in thyroid follicles in the nasopharynx of 96 hpf embryos. Treatment with exogenous triiodothyronine (T3) and T4 improved, but did not completely rescue, PTU-induced neural crest defects. Thus, PTU should be used with caution when studying zebrafish embryogenesis as it alters the threshold of different signaling pathways important during craniofacial development. The effects of PTU on neural crest development are partially caused by thyroid hormone signaling

Replication protein A physically interacts with the Bloom's syndrome protein and stimulates its helicase activity.

Bloom's syndrome is a rare autosomal recessive disorder characterized by genomic instability and predisposition to cancer. BLM, the gene defective in Bloom's syndrome, encodes a 159-kDa protein possessing DNA-stimulated ATPase and ATP-dependent DNA helicase activities. We have examined mechanistic aspects of the catalytic functions of purified recombinant BLM protein. Through analyzing the effects of different lengths of DNA cofactor on ATPase activity, we provide evidence to suggest that BLM translocates along single-stranded DNA in a processive manner. The helicase reaction catalyzed by BLM protein was examined as a function of duplex DNA length. We show that BLM catalyzes unwinding of short DNA duplexes (/=259-bp). The presence of the human single-stranded DNA-binding protein (human replication protein A (hRPA)) stimulates the BLM unwinding reaction on the 259-bp partial duplex DNA substrate. Heterologous single-stranded DNA-binding proteins fail to stimulate similarly the helicase activity of BLM protein. This is the first demonstration of a functional interaction between BLM and another protein. Consistent with a functional interaction between hRPA and the BLM helicase, we demonstrate a direct physical interaction between the two proteins mediated by the 70-kDa subunit of RPA. The interactions between BLM and hRPA suggest that the two proteins function together in vivo to unwind DNA duplexes during replication, recombination, or repair

Cryo-Electron Tomography of Marburg Virus Particles and Their Morphogenesis within Infected Cells

Ultrastructural analysis of a filovirus assembling within infected eukaryotic cells reveals differences in structure and assembly mechanisms between related RNA viruses

The distinct roles of the nucleus and nucleus-cytoskeleton connections in three-dimensional cell migration

Cells often migrate in vivo in an extracellular matrix that is intrinsically three-dimensional (3D) and the role of actin filament architecture in 3D cell migration is less well understood. Here we show that, while recently identified linkers of nucleoskeleton to cytoskeleton (LINC) complexes play a minimal role in conventional 2D migration, they play a critical role in regulating the organization of a subset of actin filament bundles – the perinuclear actin cap - connected to the nucleus through Nesprin2giant and Nesprin3 in cells in 3D collagen I matrix. Actin cap fibers prolong the nucleus and mediate the formation of pseudopodial protrusions, which drive matrix traction and 3D cell migration. Disruption of LINC complexes disorganizes the actin cap, which impairs 3D cell migration. A simple mechanical model explains why LINC complexes and the perinuclear actin cap are essential in 3D migration by providing mechanical support to the formation of pseudopodial protrusions

Synthetic Biology: Mapping the Scientific Landscape

This article uses data from Thomson Reuters Web of Science to map and analyse the scientific landscape for synthetic biology. The article draws on recent advances in data visualisation and analytics with the aim of informing upcoming international policy debates on the governance of synthetic biology by the Subsidiary Body on Scientific, Technical and Technological Advice (SBSTTA) of the United Nations Convention on Biological Diversity. We use mapping techniques to identify how synthetic biology can best be understood and the range of institutions, researchers and funding agencies involved. Debates under the Convention are likely to focus on a possible moratorium on the field release of synthetic organisms, cells or genomes. Based on the empirical evidence we propose that guidance could be provided to funding agencies to respect the letter and spirit of the Convention on Biological Diversity in making research investments. Building on the recommendations of the United States Presidential Commission for the Study of Bioethical Issues we demonstrate that it is possible to promote independent and transparent monitoring of developments in synthetic biology using modern information tools. In particular, public and policy understanding and engagement with synthetic biology can be enhanced through the use of online interactive tools. As a step forward in this process we make existing data on the scientific literature on synthetic biology available in an online interactive workbook so that researchers, policy makers and civil society can explore the data and draw conclusions for themselves

Distinguishing the Impacts of Inadequate Prey and Vessel Traffic on an Endangered Killer Whale (Orcinus orca) Population

Managing endangered species often involves evaluating the relative impacts of multiple anthropogenic and ecological pressures. This challenge is particularly formidable for cetaceans, which spend the majority of their time underwater. Noninvasive physiological approaches can be especially informative in this regard. We used a combination of fecal thyroid (T3) and glucocorticoid (GC) hormone measures to assess two threats influencing the endangered southern resident killer whales (SRKW; Orcinus orca) that frequent the inland waters of British Columbia, Canada and Washington, U.S.A. Glucocorticoids increase in response to nutritional and psychological stress, whereas thyroid hormone declines in response to nutritional stress but is unaffected by psychological stress. The inadequate prey hypothesis argues that the killer whales have become prey limited due to reductions of their dominant prey, Chinook salmon (Oncorhynchus tshawytscha). The vessel impact hypothesis argues that high numbers of vessels in close proximity to the whales cause disturbance via psychological stress and/or impaired foraging ability. The GC and T3 measures supported the inadequate prey hypothesis. In particular, GC concentrations were negatively correlated with short-term changes in prey availability. Whereas, T3 concentrations varied by date and year in a manner that corresponded with more long-term prey availability. Physiological correlations with prey overshadowed any impacts of vessels since GCs were lowest during the peak in vessel abundance, which also coincided with the peak in salmon availability. Our results suggest that identification and recovery of strategic salmon populations in the SRKW diet are important to effectively promote SRKW recovery