Accessing NCBI data using the NCBI Sequence Viewer and Genome Data Viewer (GDV).

The National Center for Biotechnology Information (NCBI) is an archive providing free access to a wide range and large volume of biological sequence data and literature. Staff scientists at NCBI analyze user-submitted data in the archive, producing gene and SNP annotation and generating sequence alignment tools. NCBI's flagship genome browser, Genome Data Viewer (GDV), displays our in-house RefSeq annotation; is integrated with other NCBI resources such as Gene, dbGaP, and BLAST; and provides a platform for customized analysis and visualization. Here, we describe how members of the biomedical research community can use GDV and the related NCBI Sequence Viewer (SV) to access, analyze, and disseminate NCBI and custom biomedical sequence data. In addition, we report how users can add SV to their own web pages to create a custom graphical sequence display without the need for infrastructure investments or back-end deployments.

Fibroblast growth factor 1 (FGFR1) modulation regulates repair capacity of oligodendrocyte progenitor cells following chronic demyelination.

The adult mammalian brain contains multiple populations of endogenous progenitor cell types. However, following CNS trauma or disease, the regenerative capacity of progenitor populations is typically insufficient and may actually be limited by non-permissive or inhibitory signals in the damaged parenchyma. Remyelination is the most effective and simplest regenerative process in the adult CNS yet is still insufficient following repeated or chronic demyelination. Our previous in vitro studies demonstrated that fibroblast growth factor receptor 1 (FGFR1) signaling inhibited oligodendrocyte progenitor (OP) differentiation into mature oligodendrocytes. Therefore, we questioned whether FGFR1 signaling may inhibit the capacity of OP cells to generate oligodendrocytes in a demyelinating disease model and whether genetically reducing FGFR1 signaling in oligodendrocyte lineage cells could enhance the capacity for remyelination. FGFR1 was found to be upregulated in the corpus callosum during cuprizone mediated demyelination and expressed on OP cells just prior to remyelination. Plp/CreER(T):Fgfr1(fl/fl) mice were administered tamoxifen to induce conditional Fgfr1 deletion in oligodendrocyte lineage cells. Tamoxifen administration during chronic demyelination resulted in reduced FGFR1 expression in OP cells. OP proliferation and population size were not altered one week after tamoxifen treatment. Tamoxifen was then administered during chronic demyelination and mice were given a six week recovery period without cuprizone in the chow. After the recovery period, OP numbers were reduced and the number of mature oligodendrocytes was increased, indicating an effect of FGFR1 reduction on OP differentiation. Importantly, tamoxifen administration in Plp/CreER(T):Fgfr1(fl/fl) mice significantly promoted remyelination and axon integrity. These results demonstrate a direct effect of FGFR1 signaling in oligodendrocyte lineage cells as inhibiting the repair capacity of OP cells following chronic demyelination in the adult CNS.

Pharmacological strategies for the regulation of inducible nitric oxide synthase: neurodegenerative versus neuroprotective mechanisms.

Inducible nitric oxide synthase (iNOS) is one of three NOS isoforms generating nitric oxide (NO) by the conversion of l-arginine to l-citrulline. iNOS has been found to be a major contributor to initiation/exacerbation of the central nervous system (CNS) inflammatory/degenerative conditions through the production of excessive NO which generates reactive nitrogen species (RNSs). Activation of iNOS and NO generation has come to be accepted as a marker and therapeutic target in neuroinflammatory conditions such as those observed in ischemia, multiple sclerosis (MS), spinal cord injury (SCI), Alzheimer's disease (AD), and inherited peroxisomal (e.g. X-linked adrenoleukodystrophy; X-ALD) and lysosomal disorders (e.g. Krabbe's disease). However, with the emergence of reports on the neuroprotective facets of NO, the prior dogma about NO being solely detrimental has had to be modified. While RNSs such as peroxynitrite (ONOO(-)) have been linked to lipid peroxidation, neuronal/oligodendrocyte loss, and demyelination in neurodegenerative diseases, limited NO generation by GSNO has been found to promote vasodilation and attenuate vascular injury under the same ischemic conditions. NO generated from GSNO acts as second messenger molecular which through S-nitrosylation has been shown to control important cellular processes by regulation of expression/activity of certain proteins such as NF-kappaB. It is now believed that the environment and the context in which NO is produced largely determines the actions (good or bad) of this molecule. These multi-faceted aspects of NO make therapeutic interference with iNOS activity even more complicated since complete ablation of iNOS activity has been found to be rather more detrimental than protective in most neurodegenerative conditions. Investigators in search of iNOS modulating pharmacological agents have realized the need of a delicate balance so as to allow the production of physiologically relevant amounts of NO (such as those required for host defence/neutotransmission/vasodilation, etc.) but at the same time block the generation of RNSs through repressing excessive NO levels (such as those causing neuronal/tissue damage and demyelination, etc.). The past years have seen a noteworthy increase in novel agents that might prove useful in achieving the aim of harnessing the good and blocking the undesirable actions of NO. It is the aim of this review to provide basic insights into the NOS family of enzymes with special emphasis of the role of iNOS in the CNS, in the first part. In the second part of the review, we will strive to provide an exhaustive compilation of the prevalent strategies being tested for the therapeutic modulation of iNOS and NO production.

A novel role of lactosylceramide in the regulation of lipopolysaccharide/interferon-gamma-mediated inducible nitric oxide synthase gene expression: implications for neuroinflammatory diseases.

In the present study a possible role of glycosphingolipids (GSLs) in inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production after spinal cord injury (SCI) in rats has been established. In primary rat astrocytes lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) treatment increased the intracellular levels of lactosylceramide (LacCer) and induced iNOS gene expression. d-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCI (PDMP), a glucosylceramide synthase and LacCer synthase (galactosyltransferase, GalT-2) inhibitor, inhibited LPS/IFN-gamma induced iNOS expression, which was reversed by exogenously supplied LacCer, but not by other glycosphingolipids. LPS/IFN-gamma caused a rapid increase in the activity of GalT-2 and synthesis of LacCer. Silencing of GalT-2 gene with the use of antisense oligonucleotides resulted in decreased LPS/IFN-gamma-induced iNOS, TNF-alpha, and IL-1beta gene expression. The PDMP-mediated reduction in LacCer production and inhibition of iNOS expression correlated with decreased Ras and ERK1/2 activation along with decreased IkappaB phosphorylation, NF-kappaB DNA binding activity, and NF-kappaB-luciferase reporter activity. LacCer-mediated Ras activation was redox-mediated and was attenuated by antioxidants N-acetyl cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). In vivo administration of PDMP after SCI resulted in improved functional outcome (Basso, Beattie, Bresnahan score); inhibition of iNOS, TNF-alpha, and IL-1beta expression; decreased neuronal apoptosis; and decreased tissue necrosis and demyelination. The in vivo studies supported the conclusions drawn from cell culture studies and provided evidence for the possible role of GalT-2 and LacCer in SCI-induced inflammation and pathology. To our knowledge this is the first report of a role of LacCer in iNOS expression and the advantage of GSL depletion in attenuating post-SCI inflammation to improve the outcome of SCI.

Quantifying the effects of soil temperature, moisture and sterilization on elemental mercury formation in boreal soils.

Soils are a source of elemental mercury (Hg(0)) to the atmosphere, however the effects of soil temperature and moisture on Hg(0) formation is not well defined. This research quantifies the effect of varying soil temperature (278-303 K), moisture (15-80% water filled pore space (WFPS)) and sterilization on the kinetics of Hg(0) formation in forested soils of Nova Scotia, Canada. Both, the logarithm of cumulative mass of Hg(0) formed in soils and the reduction rate constants (k values) increased with temperature and moisture respectively. Sterilizing soils significantly (p < 0.05, n = 10) decreased the percent of total Hg reduced to Hg(0). We describe the fundamentals of Hg(0) formation in soils and our results highlight two key processes: (i) a fast abiotic process that peaks at 45% WFPS and depletes a small pool of Hg(0) and; (ii) a slower, rate limiting biotic process that generates a large pool of reducible Hg(II).

Cuprizone demyelination of the corpus callosum in mice correlates with altered social interaction and impaired bilateral sensorimotor coordination.

For studies of remyelination in demyelinating diseases, the cuprizone model of CC (corpus callosum) demyelination has experimental advantages that include overall size, proximity to neural stem cells of the subventricular zone, and correlation with a lesion predilection site in multiple sclerosis. In addition, cuprizone treatment can be ended to allow more direct analysis of remyelination than with viral or autoimmune models. However, CC demyelination lacks a useful functional correlate in rodents for longitudinal analysis throughout the course of demyelination and remyelination. In the present study, we tested two distinct behavioural measurements in mice fed 0.2% cuprizone. Running on a 'complex' wheel with varied rung intervals requires integration between cerebral hemispheres for rapid bilateral sensorimotor coordination. Maximum running velocity on the 'complex' wheel decreased during acute (6 week) and chronic (12 week) cuprizone demyelination. Running velocity on the complex wheel distinguished treated (for 6 weeks) from non-treated mice, even after a 6-week recovery period for spontaneous remyelination. A second behavioural assessment was a resident-intruder test of social interaction. The frequency of interactive behaviours increased among resident mice after acute or chronic demyelination. Differences in both sensorimotor coordination and social interaction correlated with demonstrated CC demyelination. The wheel assay is applicable for longitudinal studies. The resident-intruder assay provides a complementary assessment of a distinct modality at a specific time point. These behavioural measurements are sufficiently robust for small cohorts as a non-invasive assessment of demyelination to facilitate analysis of subsequent remyelination. These measurements may also identify CC involvement in other mouse models of central nervous system injuries and disorders.

Post-trauma Lipitor treatment prevents endothelial dysfunction, facilitates neuroprotection, and promotes locomotor recovery following spinal cord injury.

We have previously reported neuroprotection in spinal cord injury (SCI) by Lipitor [atorvastatin (AT)]-pre-treatment. Though informative, pre-treatment studies find only limited clinical application as trauma occurrence is unpredictable. Therefore, this study investigates the efficacy of AT treatment post-SCI. In a rat model of contusion-SCI resulting in complete hindlimb paralysis, AT treatment (5 mg/kg; gavage) was begun 2, 4, or 6 h post-SCI followed by a once daily dose thereafter for 6 weeks. While the placebo vehicle (VHC)-SCI rats showed substantial functional deficit, AT-SCI animals exhibited significant functional recovery. AT diminished injury-induced blood-spinal cord barrier (BSCB) dysfunction with significantly reduced infiltration and tumor necrosis factor-alpha/interleukin-1beta/inducible nitric oxide synthase expression at site of injury. BSCB protection in AT-SCI was attributable to attenuated matrix metalloproteinase-9 (MMP9) expression - a central player in BSCB disruption. Furthermore, endothelial MMP9 expression was found to be RhoA/ROCK pathway-mediated and regulated by AT through an isoprenoid-dependent mechanism. Attenuation of these early inflammatory events reduced secondary damage. Significant reduction in axonal degeneration, myelin degradation, gliosis, and neuronal apoptosis with resultant enhancement in tissue sparing was observed in AT-SCI compared with VHC-SCI. In summary, this novel report presenting the efficacy of post-injury AT treatment might be of critical therapeutic value as effective treatments are currently unavailable for SCI.

Complement plays an important role in spinal cord injury and represents a therapeutic target for improving recovery following trauma.

Initiation of an inflammatory cascade following traumatic spinal cord injury (SCI) is thought to cause secondary injury and to adversely impact functional recovery, although the mechanisms involved are not well defined. We report on the dynamics of complement activation and deposition in the mouse spinal cord following traumatic injury, the role of complement in the development of SCI, and the characterization of a novel targeted complement inhibitor. Following traumatic injury, mice deficient in C3 had a significantly improved locomotor score when compared with wild-type controls, and analysis of their spinal cords revealed significantly more tissue sparing, with significantly less necrosis, demyelination, and neutrophil infiltration. Wild-type mice were also treated with CR2-Crry, a novel inhibitor of complement activation that targets to sites of C3 deposition. A single intravenous injection of CR2-Crry 1 hour after traumatic injury improved functional outcome and pathology to an extent similar to that seen in C3-deficient animals. CR2-Crry specifically targeted to the injured spinal cord in a distribution pattern corresponding to that seen for deposited C3. As immunosuppression is undesirable in patients following SCI, targeted CR2-Crry may provide appropriate bioavailability to treat SCI at a dose that does not significantly affect systemic levels of serum complement activity.

A novel role of lactosylceramide in the regulation of tumor necrosis factor alpha-mediated proliferation of rat primary astrocytes. Implications for astrogliosis following neurotrauma.

The present study describes the role of glycosphingolipids in neuroinflammatory disease and investigates tumor necrosis factor alpha (TNFalpha)-induced astrogliosis following spinal cord injury. Astrogliosis is the hallmark of neuroinflammation and is characterized by proliferation of astrocytes and increased glial fibrillary acidic protein (GFAP) gene expression. In primary astrocytes, TNFalpha stimulation increased the intracellular levels of lactosylceramide (LacCer) and induced GFAP expression and astrocyte proliferation. D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCl (PDMP), a glucosylceramide synthase and LacCer synthase (GalT-2) inhibitor, inhibited astrocyte proliferation and GFAP expression, which were reversed by exogenous supplementation of LacCer but not by other glycosphingolipids. TNFalpha caused a rapid increase in the activity of GalT-2 and synthesis of LacCer. Silencing of GalT-2 gene using antisense oligonucleotides also attenuated the proliferation of astrocytes and GFAP expression. The PDMP and antisense-mediated inhibition of proliferation and GFAP expression was well correlated with decreased Ras/ERK1/2 pathway activation. Furthermore, TNFalpha-mediated astrocyte proliferation and GFAP expression was also inhibited by LY294002, a phosphatidylinositol 3-kinase inhibitor, which was reversed by exogenous LacCer. LY294002 also inhibited TNFalpha-induced GalT-2 activation and LacCer synthesis, suggesting a phosphatidylinositol 3-kinase-mediated regulation of GalT-2. In vivo, PDMP treatment attenuated chronic ERK1/2 activation and spinal cord injury (SCI)-induced astrocyte proliferation with improved functional recovery post-SCI. Therefore, the in vivo studies support the conclusions drawn from cell culture studies and provide evidence for the role of LacCer in TNFalpha-induced astrogliosis in a rat model of SCI. To our knowledge, this is the first report demonstrating the role of LacCer in the regulation of TNFalpha-induced proliferation and reactivity of primary astrocytes.

Attenuation of acute inflammatory response by atorvastatin after spinal cord injury in rats.

Spinal cord injury (SCI) is a devastating and complex clinical condition involving proinflammatory cytokines and nitric oxide toxicity that produces a predictable pattern of progressive injury entailing neuronal loss, axonal destruction, and demyelination at the site of impact. The involvement of proinflammatory cytokines and inducible nitric oxide synthase (iNOS) in exacerbation of SCI pathology is well documented. We have reported previously the antiinflammatory properties and immunomodulatory activities of statins (3-hydroxy-3-methylglutaryl [HMG]-CoA reductase inhibitors) in the animal model of multiple sclerosis, experimental allergic encephalitis (EAE). The present study was undertaken to investigate the efficacy of atorvastatin (Lipitor; LP) treatment in attenuating SCI-induced pathology. Immunohistochemical detection and real-time PCR analysis showed increased expression of iNOS, tumor necrosis factor alpha (TNFalpha) and interleukin 1beta (IL-1beta) after SCI. In addition, neuronal apoptosis was detected 24 hr after injury followed by a profound increase in ED1-positive inflammatory infiltrates, glial fibrillary acidic protein (GFAP)-positive reactive astrocytes, and oligodendrocyte apoptosis by 1 week after SCI relative to control. LP treatment attenuated the SCI-induced iNOS, TNFalpha, and IL-1beta expression. LP also provided protection against SCI-induced tissue necrosis, neuronal and oligodendrocyte apoptosis, demyelination, and reactive gliosis. Furthermore, rats treated with LP scored much higher on the locomotor rating scale after SCI (19.13 +/- 0.53) than did untreated rats (9.04 +/- 1.22). This study therefore reports the beneficial effect of atorvastatin for the treatment of SCI-related pathology and disability.