Glycogen synthase kinase 3 has a limited role in cell cycle regulation of cyclin D1 levels

BACKGROUND: The expression level of cyclin D1 plays a vital role in the control of proliferation. This protein is reported to be degraded following phosphorylation by glycogen synthase kinase 3 (GSK3) on Thr-286. We recently showed that phosphorylation of Thr-286 is responsible for a decline in cyclin D1 levels during S phase, an event required for efficient DNA synthesis. These studies were undertaken to test the possibility that phosphorylation by GSK3 is responsible for the S phase specific decline in cyclin D1 levels, and that this event is regulated by the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway which controls GSK3. RESULTS: We found, however, that neither PI3K, AKT, GSK3, nor proliferative signaling activity in general is responsible for the S phase decline in cyclin D1 levels. In fact, the activity of these signaling kinases does not vary through the cell cycle of proliferating cells. Moreover, we found that GSK3 activity has little influence over cyclin D1 expression levels during any cell cycle phase. Inhibition of GSK3 activity by siRNA, LiCl, or other chemical inhibitors failed to influence cyclin D1 phosphorylation on Thr-286, even though LiCl efficiently blocked phosphorylation of β-catenin, a known substrate of GSK3. Likewise, the expression of a constitutively active GSK3 mutant protein failed to influence cyclin D1 phosphorylation or total protein expression level. CONCLUSION: Because we were unable to identify any proliferative signaling molecule or pathway which is regulated through the cell cycle, or which is able to influence cyclin D1 levels, we conclude that the suppression of cyclin D1 levels during S phase is regulated by cell cycle position rather than signaling activity. We propose that this mechanism guarantees the decline in cyclin D1 levels during each S phase; and that in so doing it reduces the likelihood that simple over expression of cyclin D1 can lead to uncontrolled cell growth

Rabies Management Implications Based on Raccoon Population Density Indexes

An estimate or index of target species density is important in determining oral rabies vaccination (ORV) bait densities to control and eliminate specific rabies variants. From 1997–2011, we indexed raccoon (Procyon lotor) densities 253 times based on cumulative captures on 163 sites from Maine to Alabama, USA, near ORV zones created to prevent raccoon rabies from spreading to new areas. We conducted indexing under a common cage trapping protocol near the time of annual ORV to aid in bait density decisions. Unique raccoons (n = 8,415) accounted for 68.0% of captures (n = 12,367). We recaptured raccoons 2,669 times. We applied Schnabel and Huggins mark‐recapture models on sites with ≥3 years of capture data and ≥25% recaptures as context for raccoon density indexes (RDIs). Simple linear relationships between RDIs and mark‐recapture estimates supported application of our 2 index. Raccoon density indexes ranged from 0.0–56.9 raccoons/km . For bait density decisions, we evaluated RDIs in the following 4 raccoon density groups, which were statistically different: (0.0–5.0 [n = 70], 5.1–15.0 [n = 129], 15.1–25.0 [n = 31], and \u3e25.0 raccoons/km2 [n = 23]). Mean RDI was positively associated with a higher percentage of developed land cover and a lower percentage of evergreen forest. Non‐target species composition (excluding recaptured raccoons) accounted for 32.0% of captures. Potential bait competitors accounted for 76.5% of non‐targets. The opossum (Didelphis virginiana) was the primary potential bait competitor from 27°N to 44°N latitude, north of which it was numerically replaced by the striped skunk (Mephitis mephitis). We selected the RDI approach over mark-recapture methods because of costs, geographic scope, staff availability, and the need for supplemental serologic samples. The 4 density groups provided adequate sensitivity to support bait density decisions for the current 2 bait density options. Future improvements to the method include providing random trapping locations to field personnel to prevent trap clustering and marking non‐targets to better characterize bait competitors

Preface

Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of Elsevier for personal use, not for redistribution. The definitive version was published in Deep Sea Research Part II: Topical Studies in Oceanography 103 (2014): 1-5, doi:10.1016/j.dsr2.2014.02.007.The Gulf of Maine (GOM) is a continental shelf sea in the northwest Atlantic, USA that supports highly-productive shellfisheries that are frequently contaminated by toxigenic Alexandrium fundyense blooms and outbreaks of paralytic shellfish poisoning (PSP), resulting in significant economic and social impacts. Additionally, an emerging threat to these resources is from blooms of toxic Pseudo-nitzschia species that produce domoic acid, the toxin responsible for amnesic shellfish poisoning (ASP). Nearshore shellfish toxins are monitored by state agencies, whereas most offshore stocks have had little or no routine monitoring. As a result, large areas of federal waters have been indefinitely closed or their shellfish beds underexploited because of the potential risk these toxins pose and the lack of scientific understanding and management tools. Patterns and dynamics of Alexandrium blooms and the resulting shellfish toxicity in nearshore waters were examined in a number of research projects, the largest being the Ecology and Oceanography of Harmful Algal Blooms (ECOHAB)-Gulf of Maine (GOM), a five-year regional program emphasizing field surveys, laboratory studies and numerical modeling. At the completion of the ECOHAB-GOM program (documented in Anderson et al., 2005), great progress was made in understanding A. fundyense blooms and resulting shellfish toxicity in nearshore waters, but there were major unknowns that still required investigation. For example, little was known about A. fundyense bloom dynamics in the waters south and east of Cape Cod, Massachusetts, and in particular, about the link between blooms in surface waters and toxicity in deep offshore shellfish. Large areas of offshore shellfish beds were off limits to harvest, including a 40,000 km2 region closed during the 2005 bloom and a much larger zone (~80,000 km2) including portions of Georges Bank was closed in 1990 after high levels of PSP toxicity were detected. In recent years, pressures were mounting from industry to open those offshore areas and to develop management strategies so that surfclam (Spisula solidissima), ocean quahog (Arctica islandica), and roe-on sea scallop (Placopecten magellanicus) fisheries could be opened. In response to these unknowns and societal needs, a new multi-investigator program, GOMTOX (Gulf of Maine Toxicity), was formulated and ultimately funded through the NOAA ECOHAB program. GOMTOX was a regional observation and modeling program that investigated the patterns and mechanisms underlying A. fundyense and Pseudo-nitzschia blooms and the resulting toxicity in shellfish in the southern GOM and its adjacent New England shelf waters, with special emphasis on the delivery pathways, mechanisms, and dynamics of offshore shellfish toxicity. The GOMTOX team of investigators included 16 principal investigators from eight institutions and, continuing in the ECOHAB-GOM tradition, strong participation from federal and state resource managers as well as representatives of the shellfish industry. This team worked together for over five years, running numerous large-scale survey cruises of Alexandrium cells and cysts, and also supporting industry cruises to collect shellfish from offshore sites including Georges Bank. Other efforts included participation in National Marine Fisheries Service surveys for shellfish (sea scallops, surfclams, and ocean quahogs), numerical modeling studies, deployment of sediment traps, and laboratory and ship-based experiments to investigate grazing and other processes that might regulate blooms and deliver toxins to shellfish in deeper waters. A smaller-scale but concurrent effort collected samples to characterize Pseudo-nitzschia species and their potential toxicity in the region.We gratefully acknowledge the support of NOAA through the ECOHAB program. Partial support for some of the studies contained herein was provided by NSF and NIEHS through the Woods Hole Center for Oceans and Human Health. Funding for J.L. Martin’s contributions from the Bay of Fundy was provided by Fisheries and Oceans Canada and NERACOOS, which is a part of the U.S. Integrated Ocean Observing System, funded in part by National Oceanic and Atmospheric Administration (NOAA)

Decorin Expression, Straw-Like Structure, and Differentiation of Human Costal Cartilage

Costal cartilage is much understudied compared with the load-bearing cartilages. Abnormally grown costal cartilages are associated with the inherited chest wall deformities pectus excavatum and pectus carinatum resulting in sunken and pigeon chests, respectively. A lack of understanding of the ultrastructural and molecular biology of costal cartilage is a major confounder in predicting causes and outcomes of these disorders. This study analyzed the structure of marginal human costal cartilage (ribs 6-10) through scanning electron and atomic force microscopes and identified the presence of straw-like structures running longitudinally. We also demonstrated that chondrocytes tend to occur singly or as doublets and that centrally located cells produce high levels of aggrecan compared with more peripherally located cells measured using immunohistochemistry. Gene expression from mRNA extracted from cartilage showed high levels of decorin expression, likely associated with the large, complex tubular structures running through this cartilage type. COL2A1, ACAN, and TIMP1 also showed higher levels of expression compared with ACTB. Analysis of gene expression ratios demonstrate that costal cartilage is under differentiated compared with published ratios for articular cartilage, likely due to the vastly different biomechanical environments of each cartilage type. Further studies need to establish whether findings described here from the costal margins are significantly different than the cartilage of the true ribs and how these values change with age

Characterization of white spot lesions formed on human enamel under microcosm biofilm for different experimental periods

The initial characteristics of white spot lesion (WSLs), such as the degree of integrated mineral loss (ΔZ), depth and pattern of mineral distribution, have an impact on further demineralization and remineralization. However, these lesion parameters have not been evaluated in WSLs produced from microcosm biofilms. Objective: This study characterized artificial white spot lesions produced on human enamel under microcosm biofilm for different experimental periods. Methodology: In total, 100 human enamel specimens (4x4mm) were assigned to 5 distinct groups (n=20/group) differing according to the period of biofilm formation (2, 4, 6, 8 or 10 days). Microcosm biofilm was produced on the specimens from a mixture of human and McBain saliva at the first 8h. Enamel samples were then exposed to McBain saliva containing 0.2% sucrose. WSLs formed were characterized by quantitative light-induced fluorescence (QLF) and transverse microradiography (TMR). Data were analyzed by ANOVA/Tukey or Kruskal-Wallis/Dunn tests (p<0.05). Results: A clear time-response pattern was observed for both analyses, but TMR was able to better discriminate among the lesions. Regarding QLF analysis, median (95%CI; %) changes in fluorescence ∆Z were -7.74(-7.74:-6.45)a, -8.52(-8.75:-8.00)ab, -9.17(-10.00:-8.71)bc, -9.58(-10.53:-8.99)bc and -10.01(-11.44:-9.72)c for 2, 4, 6, 8, and 10 days, respectively. For TMR, median (95%CI; vol%.µm) ∆Z were 1410(1299-1479)a, 2420(2327-2604)ab, 2775(2573-2899)bc, 3305(3192-3406)cd and 4330(3972-4465)d, whereas mean (SD; µm) lesion depth were 53.7(12.3)a, 71.4(12.0)a, 103.8(24.8)b, 130.5(27.2)bc, 167.2(39.3)c for 2, 4, 6, 8 and 10 days, respectively. Conclusion: The progression of WSLs formed on human enamel under microcosm biofilm can be characterized over 2-10 days, both by QLF and TMR analyses, although the latter provides better discrimination among the lesions

Cathepsin S is the major activator of the psoriasis-associated proinflammatory cytokine IL-36γ

The pro-inflammatory cytokine IL-36γ is highly expressed in epithelial cells and is a pivotal mediator of epithelial inflammation. In particular, IL-36γ is strongly associated with the inflammatory skin disease psoriasis. As with other IL-1 cytokines, IL-36γ is expressed as an inactive precursor and must be processed by specific proteases to become bioactive. Our aim therefore was to identify protease/s capable of IL-36γ activation and explore the importance of this activation in psoriasis. Using a keratinocyte-based activity assay in conjunction with small-molecule inhibitors and siRNA gene silencing, cathepsin S was identified as the major IL-36γ-activating protease expressed by epithelial cells. Interestingly, cathepsin S activity was strongly upregulated in samples extracted from psoriasis patients, relative to healthy controls. In addition, IL-36γ-Ser18, identified as the main product of cathepsin S-dependent IL-36γ cleavage, induced psoriasiform changes in human skin-equivalent models. Together, these data provide important mechanistic insights into the activation of IL-36γ, and highlight that cathepsin S-mediated activation of IL-36γ may be important in the development of numerous IL-36γ driven pathologies, in addition to psoriasis

ISA-TAB-Nano: A Specification for Sharing Nanomaterial Research Data in Spreadsheet-based Format

BACKGROUND AND MOTIVATION: The high-throughput genomics communities have been successfully using standardized spreadsheet-based formats to capture and share data within labs and among public repositories. The nanomedicine community has yet to adopt similar standards to share the diverse and multi-dimensional types of data (including metadata) pertaining to the description and characterization of nanomaterials. Owing to the lack of standardization in representing and sharing nanomaterial data, most of the data currently shared via publications and data resources are incomplete, poorly-integrated, and not suitable for meaningful interpretation and re-use of the data. Specifically, in its current state, data cannot be effectively utilized for the development of predictive models that will inform the rational design of nanomaterials. RESULTS: We have developed a specification called ISA-TAB-Nano, which comprises four spreadsheet-based file formats for representing and integrating various types of nanomaterial data. Three file formats (Investigation, Study, and Assay files) have been adapted from the established ISA-TAB specification; while the Material file format was developed de novo to more readily describe the complexity of nanomaterials and associated small molecules. In this paper, we have discussed the main features of each file format and how to use them for sharing nanomaterial descriptions and assay metadata. CONCLUSION: The ISA-TAB-Nano file formats provide a general and flexible framework to record and integrate nanomaterial descriptions, assay data (metadata and endpoint measurements) and protocol information. Like ISA-TAB, ISA-TAB-Nano supports the use of ontology terms to promote standardized descriptions and to facilitate search and integration of the data. The ISA-TAB-Nano specification has been submitted as an ASTM work item to obtain community feedback and to provide a nanotechnology data-sharing standard for public development and adoption

Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation

As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become increasingly critical. Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated. Here, we describe the discovery of novel C > A/G > T transversion artifacts found at low allelic fractions in targeted capture data. Characteristics such as sequencer read orientation and presence in both tumor and normal samples strongly indicated a non-biological mechanism. We identified the source as oxidation of DNA during acoustic shearing in samples containing reactive contaminants from the extraction process. We show generation of 8-oxoguanine (8-oxoG) lesions during DNA shearing, present analysis tools to detect oxidation in sequencing data and suggest methods to reduce DNA oxidation through the introduction of antioxidants. Further, informatics methods are presented to confidently filter these artifacts from sequencing data sets. Though only seen in a low percentage of reads in affected samples, such artifacts could have profoundly deleterious effects on the ability to confidently call rare mutations, and eliminating other possible sources of artifacts should become a priority for the research community.National Human Genome Research Institute (U.S.) (HG03067-05