Reliability of trans-generational genetic mark-recapture (tGMR) for enumerating Pacific salmon.

As Pacific salmon (Oncorhynchus spp.) decline across much of their range, it is imperative to further develop minimally invasive tools to quantify population abundance. One such advancement, trans-generational genetic mark-recapture (tGMR), uses parentage analysis to estimate the size of wild populations. Our study examined the precision and accuracy of tGMR through a comparison to a traditional mark-recapture estimate for Chilkat River Chinook salmon (O. tshawytscha) in Southeast Alaska. We examined how adult sampling location and timing impact tGMR by comparing estimates derived using samples collected in the lower river mainstem to those using samples obtained in upriver spawning tributaries. Results indicated that tGMR estimates using a representative sample of mainstem adults were most concordant with, and 3% more precise than, the traditional mark-recapture estimate for this stock. Importantly, the timing and location of adult sampling were found to impact abundance estimates, depending on what proportion of the population dies or moves to unsampled areas between downriver and upriver sampling events. Additionally, we identified potential sources of bias in tGMR arising from violations of key assumptions using a novel individual-based modeling framework, parameterized with empirical values from the Chilkat River. Simulations demonstrated that increased reproductive success and sampling selectivity of older, larger individuals, introduced negative bias into tGMR estimates. Our individual-based model offers a customizable and accessible method to identify and quantify these biases in tGMR applications (https://github.com/swrosenbaum/tGMR_simulations). We underscore the critical role of system-specific sampling design considerations in ensuring the precision and accuracy of tGMR projects. This study validates tGMR as a potentially useful tool for improved population enumeration in semelparous species.

Improving Resident Oral Health and Adherence to a Ventilator-Associated Pneumonia Bundle in a Skilled Nursing Facility.

Ventilator-associated pneumonia (VAP) is common with mechanical ventilation. VAP bundles have improved outcomes in acute care and long-term acute care hospitals. This article reports on an implementation of a VAP bundle designed for a skilled nursing facility (SNF). The primary goal was to improve oral health, a significant factor for VAP, and improve SNF nurse adherence to the bundle. Improvements in oral health and adherence were demonstrated. Nurses had difficulty with adhering to maintaining the tracheostomy cuff seal.

Cerebrospinal fluid from patients with amyotrophic lateral sclerosis inhibits sonic hedgehog function.

Sonic hedgehog (Shh) is a morphogen essential to the developing nervous system that continues to play an important role in adult life by contributing to cell proliferation and differentiation, maintaining blood-brain barrier integrity, and being cytoprotective against oxidative and excitotoxic stress, all features of importance in amyotrophic lateral sclerosis (ALS). ALS is a fatal disease characterized by selective loss of motor neurons due to poorly understood mechanisms. Evidence indicates that Shh might play an important role in ALS, and that Shh signaling might be also adversely affected in ALS. Since little is known about the functional status of Shh pathway in patients with ALS, we therefore sought to determine whether Shh protein levels or biological activity in cerebrospinal fluid (CSF) was less in ALS patients than controls, and whether these measures could be correlated with ALS disease severity and disease progression, and with other CSF analytes of biological interest in ALS. Comparing Shh levels in the CSF of normal controls (n = 13), neurological controls (n = 12), and ALS patients (n = 9) measured by ELISA, we found that CSF Shh levels were not different between controls and ALS patients. However, when assessing Shh biological activity in CSF using in vitro cell-based assays, which measure Shh activity as inducible Gli-driven luminescence, we found that in the presence of exogenous recombinant Shh or the Shh agonist, purmorphamine, the inducible activity of CSF was significantly augmented in the control groups as expected, but not in the ALS group, suggesting the presence of an inhibitor of Shh signaling in ALS CSF samples. Since purmorphamine acts on Smoothened, downstream of Shh and its receptor Patched, the inhibitory action is downstream of Smoothened. Our results also demonstrated that while the inhibitory effect of ALS CSF on Shh signaling did not correlate significantly with ALS disease characteristics, the levels of IL-1ß and TNF-α did. In addition to being significantly elevated in ALS CSF, these cytokines negatively correlated with the disease duration, whereas GDF11 was a favorable predictor of ALS clinical score. We also found that TNF-α significantly inhibited Shh biological activity in vitro, potentially suggesting a novel role of TNF-α in ALS pathogenesis. Collectively, this is the first report demonstrating that Shh signaling in CSF of ALS patients is compromised.

Crosstalk between Notch and Sonic hedgehog signaling in a mouse model of amyotrophic lateral sclerosis.

The developmental morphogen Sonic hedgehog (Shh) may continue to play a sustaining role in adult motor neurons, of potential relevance to motor neuron diseases including amyotrophic lateral sclerosis. The Shh signaling pathway is incompletely understood and interactions with other signaling pathways are possible. We focus here on Notch, and first show that there is an almost linear reduction in light output from a Gli reporter in Shh Light II cells in the presence of increasing concentrations of the Notch inhibitor DAPT (r=0.982). Second, in the spinal cord of mutant superoxide dismutase mice, but not control mice, a key marker of Notch signaling changes with age. Before the onset of clinical signs, the Notch intracellular domain is expressed predominantly in motor neurons, but by 125 days of age, Notch intracellular domain expression is markedly reduced in motor neurons and increased in neighboring astroglia. Third, there is a parallel reduction in Gli protein expression in mutant superoxide dismutase mouse spinal motor neurons, consistent with the observed reduction in Notch signaling and also a redistribution of Gli away from the nucleus. Thus, there is a reduction in motor neuronal Notch signaling and associated changes in Shh signaling, occurring coincidentally with disease expression, that may contribute toward the dysfunction and death of motor neurons in amyotrophic lateral sclerosis.

Trophic and proliferative effects of Shh on motor neurons in embryonic spinal cord culture from wildtype and G93A SOD1 mice.

BACKGROUND: The developmental morphogen sonic hedgehog (Shh) may continue to play a trophic role in the support of terminally-differentiated motor neurons, of potential relevance to motor neuron disease. In addition, it may support the proliferation and differentiation of endogenous stem cells along motor neuronal lineages. As such, we have examined the trophic and proliferative effects of Shh supplementation or Shh antagonism in embryonic spinal cord cell cultures derived from wildtype or G93A SOD1 mice, a mouse model of amyotrophic lateral sclerosis. RESULTS: Shh supported survival, and stimulated growth of motor neurons, neurite outgrowth, and neurosphere formation in primary culture derived from both G93A SOD1 and WT mice. Shh increased the percentage of ciliated motor neurons, especially in G93A SOD1 culture. Shh-treated cultures showed increased neuronal proliferation compared to controls and especially cyclopamine treated cultures, from G93A SOD1 and WT mice. Moreover, Shh enhanced cell survival and differentiation of motor neuron precursors in WT culture. CONCLUSIONS: Shh is neurotrophic to motor neurons and has mitogenic effects in WT and mSOD1 G93A culture in vitro.

Sonic hedgehog is cytoprotective against oxidative challenge in a cellular model of amyotrophic lateral sclerosis.

We have previously demonstrated that primary cilia on spinal motor neurons are reduced in G93A SOD1 (mSOD) mice, a mouse model of amyotrophic lateral sclerosis (ALS). Sonic hedgehog (Shh) signaling involves the primary cilium and Shh has been shown to be cytoprotective in models of other neurodegenerative diseases. Thus, the Shh signaling pathway may bear further study in ALS. Accordingly, we established that interference with the Shh pathway (with the Shh antagonist cyclopamine or with miRNA 3245p) sensitized HT22 cells, while augmentation of the Shh pathway (with Shh or the Shh agonist purmorphamine) protected cells against hydrogen peroxide (H2O2) challenge. We ectopically expressed mSOD, human wild-type SOD1 (wtSOD), or an empty vector in HT22 cells. Compared to empty vector, wtSOD decreased cell death and mSOD increased cell death in response to H2O2 challenge. Treatment with cyclopamine or miRNA 3245p sensitized all three transfections to H2O2 challenge. Treatment with recombinant human Shh or purmorphamine decreased cell death after H2O2 challenge, an effect more pronounced in mSOD cells. Compared with empty vector, overexpression of wtSOD increased Shh and Gli transcript levels and increased activity in a Gli-responsive reporter assay. Overexpression of mSOD did not change Shh transcript levels, but decreased Gli transcript levels, especially Gli3, and reduced activity in a Gli reporter assay. These results suggest that overexpression of mSOD but not wtSOD reduces signaling in the Shh pathway and renders mSOD cells more susceptible to H2O2 challenge, and that treatment with Shh or Shh agonists is cytoprotective to mSOD cells. Shh or Shh agonists merit further consideration as potential therapy in ALS.

Adenylyl cyclase type 3, a marker of primary cilia, is reduced in primary cell culture and in lumbar spinal cord in situ in G93A SOD1 mice.

BACKGROUND: The primary cilium is a solitary organelle important in cellular signaling, that projects from the cell surface of most growth-arrested or post-mitotic cells including neurons in the central nervous system. We hypothesized that primary cilial dysfunction might play a role in the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), and as a first step, report on the prevalence of primary cilial markers on cultured motor neurons from the lumbar spinal cord of embryonic wildtype (WT) and transgenic G93A SOD1 mice, and on motor neurons in situ in the lumbar spinal cord. RESULTS: At 7 days in culture there is no difference in the proportion of G93A SOD1 and WT motor neurons staining for the cilial marker ACIII. However, at 21 days there is a large relative drop in the proportion of ciliated G93A SOD1 motor neurons. In situ, at 40 days there was a slight relative drop in the proportion of ciliated motor neurons in G93A SOD1 mice. At 98 days of age there was no change in motor neuron ciliation in WT mice, but there was motor neuron loss and a large reduction in the proportion of surviving motor neurons bearing a primary cilium in G93A SOD1 mice. CONCLUSIONS: In primary culture and in situ in G93A SOD1 mice there is a large reduction in the proportion of motor neurons bearing a primary cilium.