Nesting Habitat and Behavior of Spiny Softshell Turtles Apalone Spinifera Hartwegi in the Missouri River, MT

Little is known about the nesting behavior and habitat of the western spiny softshell (Apalone spinifera hartwegi) in Montana where they are at the northern extent of their range and are a state Species of Concern. Our objective was to document nesting behavior, habitat, and timing in a 97-kilometer reach of the Missouri River. We radio-tagged 47 female turtles and attempted to locate nesting areas using telemetry, visual surveys from jet boat and on foot, and by observation from shore-based blinds. We located 27 nests; 15 were on islands, 12 were aggregated, and 2 were depredated. Nesting occurred following the peak river stage from about July 7 to July 28. Twenty-three nests were in mixed gravel and 4 nests were in sand substrates. Distance from water’s edge to the nest ranged from 1.9 m to 27 m and height of nest above the water surface elevation ranged from 0.25 m to 1.9 m. Vegetation at nest sites was sparse, ranging from 0 to 15 percent vegetative cover. Emergence of hatchlings was documented for 17 nests and occurred from about September 1 to September 20. All 17 successful nests were in gravel substrate; we did not document any emergence from nests in sand. Lack of emergence from sand nests may be related to the cumulative thermal regime in the nest chamber during the period from peak discharge until the onset of freezing in autumn. In 2012, we will investigate the thermal environment in gravel and sand nesting substrates

Nesting Ecology of Spiny Softshell Turtles on the Missouri River in Montana: Zoogeographic and Management Implications

The nesting ecology of western spiny softshell turtles (Apolone spinifera hartwegi) in Montana, where they are at the northern extent of their range and a state Species of Concern, is poorly known. We used telemetry, visual surveys, observation from shore-based blinds, and remote cameras to document nesting behavior, habitat, and timing in a 97-km reach of the Missouri River. We located 25 nests in 2011 and 97 in 2012. Most nests were in mixed-gravel substrates; only 3 percent were in pure sand. Vegetative cover at nest sites was sparse. Mean distance of nests to the water’s edge was 13.7 m and mean height above the water surface elevation was 0.7 m. Proportion of nests found on island and mainland habitats were similar in 2011, but 90 percent of nests were on islands in 2012. Predation occurred on 46 nests; mainland nests incurred higher predation rates than island nests. Nesting followed annual peak river stage, and mostly occurred in the afternoon. Durations of nesting, incubation, and emergence periods were similar in both years, but nesting and emergence occurred about three weeks later in 2011 than in 2012. Only 36 percent of nests were successful in 2011, but 60 percent were successful in 2012. Flooding in 2011 probably decreased nesting effort and success by reducing habitat availability and delaying the onset of nesting, which thereby prematurely ended incubation. However, flood events maintain and create nesting habitats by clearing vegetation and depositing substrates. Premature termination of incubation suggests that the northern range of this species is probably limited by successful incubation

Nesting Ecology of Spiny Softshell Turtles on the Missouri River in Montana: Zoogeographic and Management Implications

The nesting ecology of western spiny softshell turtles (Apolone spinifera hartwegi) in Montana, where they are at the northern extent of their range and a state Species of Concern, is poorly known. We used telemetry, visual surveys, observation from shore-based blinds, and remote cameras to document nesting behavior, habitat, and timing in a 97-km reach of the Missouri River. We located 25 nests in 2011 and 97 in 2012. Most nests were in mixed-gravel substrates; only 3 percent were in pure sand. Vegetative cover at nest sites was sparse. Mean distance of nests to the water’s edge was 13.7 m and mean height above the water surface elevation was 0.7 m. Proportion of nests found on island and mainland habitats were similar in 2011, but 90 percent of nests were on islands in 2012. Predation occurred on 46 nests; mainland nests incurred higher predation rates than island nests. Nesting followed annual peak river stage, and mostly occurred in the afternoon. Durations of nesting, incubation, and emergence periods were similar in both years, but nesting and emergence occurred about three weeks later in 2011 than in 2012. Only 36 percent of nests were successful in 2011, but 60 percent were successful in 2012. Flooding in 2011 probably decreased nesting effort and success by reducing habitat availability and delaying the onset of nesting, which thereby prematurely ended incubation. However, flood events maintain and create nesting habitats by clearing vegetation and depositing substrates. Premature termination of incubation suggests that the northern range of this species is probably limited by successful incubation

Managing Human-Habituated Bears to Enhance Survival, Habitat Effectiveness, and Public Viewing

The negative impacts on bears (Ursus spp.) from human activities associated with roads and developments are well documented. These impacts include displacement of bears from high-quality foods and habitats, diminished habitat effectiveness, and reduced survival rates. Additionally, increased public visitations to national parks accompanied with benign encounters with bears along park roads have caused more bears to habituate to the presence of people. In some contexts, habituation can predispose bears to being exposed to and rewarded by anthropogenic foods, which can also lower survival rates. The managers and staff of Yellowstone National Park located in Wyoming, Montana, and Idaho, USA, and Grand Teton National Park in northwestern Wyoming, USA have implemented several proactive strategies to mitigate the negative aspects of bear habituation. These strategies include providing park visitors with educational information on bear viewing etiquette, managing roadside viewing opportunities, installing bear-resistant infrastructure, hazing bears from developments, enforcing food and garbage storage regulations, and making human activities as predictable as possible to bears. Under the current management strategies, thousands of visitors are still able to view, photograph, and appreciate bears while visiting these parks each year. The opportunity to view bears provides a positive visitor experience and contributes millions of dollars to the local economies of park gateway communities. Positive bear viewing experiences also help build an important appreciation and conservation ethic for bears in people that visit national parks. For many years, managers were concerned about decreasing and threatened bear populations. Now more jurisdictions are facing new challenges caused by increasing bear populations. This paper highlights a successful attempt to address these issues

Retinoic Acid Promotes the Generation of Pancreatic Endocrine Progenitor Cells and Their Further Differentiation into β-Cells

The identification of secreted factors that can selectively stimulate the generation of insulin producing β-cells from stem and/or progenitor cells represent a significant step in the development of stem cell-based β-cell replacement therapy. By elucidating the molecular mechanisms that regulate the generation of β-cells during normal pancreatic development such putative factors may be identified. In the mouse, β-cells increase markedly in numbers from embryonic day (e) 14.5 and onwards, but the extra-cellular signal(s) that promotes the selective generation of β-cells at these stages remains to be identified. Here we show that the retinoic acid (RA) synthesizing enzyme Raldh1 is expressed in developing mouse and human pancreas at stages when β-cells are generated. We also provide evidence that RA induces the generation of Ngn3+ endocrine progenitor cells and stimulates their further differentiation into β-cells by activating a program of cell differentiation that recapitulates the normal temporal program of β-cell differentiation

Stable Mutated tau441 Transfected SH-SY5Y Cells as Screening Tool for Alzheimer’s Disease Drug Candidates

The role of hyperphosphorylation of the microtubule-associated protein tau in the pathological processes of several neurodegenerative diseases is becoming better understood. Consequently, development of new compounds capable of preventing tau hyperphosphorylation is an increasingly hot topic. For this reason, dependable in vitro and in vivo models that reflect tau hyperphosphorylation in human diseases are needed. In this study, we generated and validated an in vitro model appropriate to test potential curative and preventive compound effects on tau phosphorylation. For this purpose, a stably transfected SH-SY5Y cell line was constructed over-expressing mutant human tau441 (SH-SY5Y-TMHT441). Analyses of expression levels and tau phosphorylation status in untreated cells confirmed relevance to human diseases. Subsequently, the effect of different established kinase inhibitors on tau phosphorylation (e.g., residues Thr231, Thr181, and Ser396) was examined. It was shown with several methods including immunosorbent assays and mass spectrometry that the phosphorylation pattern of tau in SH-SY5Y-TMHT441 cells can be reliably modulated by these compounds, specifically targeting JNK, GSK-3, CDK1/5, and CK1. These four protein kinases are known to be involved in in vivo tau phosphorylation and are therefore authentic indicators for the suitability of this new cell culture model for tauopathies

Dual modification of Alzheimer’s disease PHF-tau protein by lysine methylation and ubiquitylation: a mass spectrometry approach

In sporadic Alzheimer’s disease (AD), neurofibrillary lesion formation is preceded by extensive post-translational modification of the microtubule associated protein tau. To identify the modification signature associated with tau lesion formation at single amino acid resolution, immunopurified paired helical filaments were isolated from AD brain and subjected to nanoflow liquid chromatography–tandem mass spectrometry analysis. The resulting spectra identified monomethylation of lysine residues as a new tau modification. The methyl-lysine was distributed among seven residues located in the projection and microtubule binding repeat regions of tau protein, with one site, K254, being a substrate for a competing lysine modification, ubiquitylation. To characterize methyl lysine content in intact tissue, hippocampal sections prepared from post mortem late-stage AD cases were subjected to double-label confocal fluorescence microscopy using anti-tau and anti-methyl lysine antibodies. Anti-methyl lysine immunoreactivity colocalized with 78 ± 13% of neurofibrillary tangles in these specimens. Together these data provide the first evidence that tau in neurofibrillary lesions is post-translationally modified by lysine methylation

Specific ion channels contribute to key elements of pathology during secondary degeneration following neurotrauma

Background: Following partial injury to the central nervous system, cells beyond the initial injury site undergo secondary degeneration, exacerbating loss of neurons, compact myelin and function. Changes in Ca 2+ flux are associated with metabolic and structural changes, but it is not yet clear how flux through specific ion channels contributes to the various pathologies. Here, partial optic nerve transection in adult female rats was used to model secondary degeneration. Treatment with combinations of three ion channel inhibitors was used as a tool to investigate which elements of oxidative and structural damage related to long term functional outcomes. The inhibitors employed were the voltage gated Ca 2+ channel inhibitor Lomerizine (Lom), the Ca 2+ permeable AMPA receptor inhibitor YM872 and the P2X 7 receptor inhibitor oxATP. Results: Following partial optic nerve transection, hyper-phosphorylation of Tau and acetylated tubulin immunoreactivity were increased, and Nogo-A immunoreactivity was decreased, indicating that axonal changes occurred acutely. All combinations of ion channel inhibitors reduced hyper-phosphorylation of Tau and increased Nogo-A immunoreactivity at day 3 after injury. However, only Lom/oxATP or all three inhibitors in combination significantly reduced acetylated tubulin immunoreactivity. Most combinations of ion channel inhibitors were effective in restoring the lengths of the paranode and the paranodal gap, indicative of the length of the node of Ranvier, following injury. However, only all three inhibitors in combination restored to normal Ankyrin G length at the node of Ranvier. Similarly, HNE immunoreactivity and loss of oligodendrocyte precursor cells were only limited by treatment with all three ion channel inhibitors in combination. Conclusions: Data indicate that inhibiting any of a range of ion channels preserves certain elements of axon and node structure and limits some oxidative damage following injury, whereas ionic flux through all three channels must be inhibited to prevent lipid peroxidation and preserve Ankyrin G distribution and OPCs

Genotype-Specific Differences between Mouse CNS Stem Cell Lines Expressing Frontotemporal Dementia Mutant or Wild Type Human Tau

Stem cell (SC) lines that capture the genetics of disease susceptibility provide new research tools. To assess the utility of mouse central nervous system (CNS) SC-containing neurosphere cultures for studying heritable neurodegenerative disease, we compared neurosphere cultures from transgenic mice that express human tau with the P301L familial frontotemporal dementia (FTD) mutation, rTg(tauP301L)4510, with those expressing comparable levels of wild type human tau, rTg(tauwt)21221. rTg(tauP301L)4510 mice express the human tauP301L variant in their forebrains and display cellular, histological, biochemical and behavioral abnormalities similar to those in human FTD, including age-dependent differences in tau phosphorylation that distinguish them from rTg(tauwt)21221 mice. We compared FTD-hallmark tau phosphorylation in neurospheres from rTg(tauP301L)4510 mice and from rTg(tauwt)21221 mice. The tau genotype-specific phosphorylation patterns in neurospheres mimicked those seen in mice, validating use of neurosphere cultures as models for studying tau phosphorylation. Genotype-specific tau phosphorylation was observed in 35 independent cell lines from individual fetuses; tau in rTg(tauP301L)4510 cultures was hypophosphorylated in comparison with rTg(tauwt)21221 as was seen in young adult mice. In addition, there were fewer human tau-expressing cells in rTg(tauP301L)4510 than in rTg(tauwt)21221 cultures. Following differentiation, neuronal filopodia-spine density was slightly greater in rTg(tauP301L)4510 than rTg(tauwt)21221 and control cultures. Together with the recapitulation of genotype-specific phosphorylation patterns, the observation that neurosphere lines maintained their cell line-specific-differences and retained SC characteristics over several passages supports the utility of SC cultures as surrogates for analysis of cellular disease mechanisms