Clinical and Scientific Challenges to Effectiveness Studies Under Coverage with Evidence Development in Alzheimer's Disease.

The Centers for Medicare and Medicaid Services (CMS) has recently issued a national coverage determination for US Food and Drug Administration (FDA)-approved anti-amyloid monoclonal antibodies (mAbs) for the treatment of Alzheimer's disease (AD) under coverage with evidence development (CED). CED schemes are complex, costly, and challenging, and often fail to achieve intended objectives because of administrative and implementation issues. AD is a heterogeneous, progressive neurodegenerative disorder with complex care pathway that additionally presents scientific challenges related to the choice of study design and methods used in evaluating CED schemes. These challenges are herein discussed. Clinical findings from the US Veterans Affairs healthcare system help inform our discussion of specific challenges to CED-required effectiveness studies in AD.

Historical contingency and the role of post-invasion evolution in alternative community states.

Historical contingency has long figured prominently in the conceptual frameworks of evolutionary biology and community ecology. Evolutionary biologists typically consider the effects of chance mutation and historical contingency in driving divergence and convergence of traits in populations, whereas ecologists instead are often interested in the role of historical contingency in community assembly and succession. Although genetic differences among individuals in populations can influence community interactions, variability among populations of the same species has received relatively little attention for its potential role in community assembly and succession. We used a community-level study of experimental evolution in two compositionally different assemblages of protists and rotifers to explore whether initial differences in species abundances among communities attributed to differences in evolutionary history, persisted as species that continued to evolve over time. In each assemblage, we observed significant convergence between two invaded treatments initially differing in evolutionary history over an observation period equal to ~40-80 generations for most species. Nonetheless, community structure failed to converge completely across all invaded treatments within an assemblage to a single structure. This suggests that whereas the species in the assemblage represent a common selective regime, differences in populations reflecting their evolutionary history can produce long-lasting transient alternative community states. In one assemblage, we also observed increasing within-treatment variability among replicate communities over time, suggesting that ecological drift may be another factor contributing to community change. Although subtle, these transient alternative states, in which communities differed in the abundance of interacting species, could nonetheless have important functional consequences, suggesting that the role of evolution in driving these states deserves greater attention.

Network topology and patch connectivity affect dynamics in experimental and model metapopulations.

Biological populations are rarely isolated in space and instead interact with others via dispersal in metapopulations. Theory predicts that network connectivity patterns can have critical effects on network robustness, as certain topologies, such as scale-free networks, are more tolerant to disturbances than other patterns. However, at present, experimental evidence of how these topologies affect population dynamics in a metapopulation framework is lacking. We used experimental metapopulations of the aquatic protist Paramecium tetraurelia to determine how network topology influences occupation patterns. We created metapopulations engineered to be comparable in linkage density, but differing in their degree distribution. We compared random networks to scale-free networks by evaluating local population occupancy and abundance throughout 18-30 protist generations. In parallel, we used simulations to explore differences in patch occupation patterns among topologies. Our experimental results highlighted the importance of the balance between dispersal and extinction in the interaction with spatial network topology. Under low dispersal conditions, random metapopulations of P. tetraurelia reached higher abundance and higher occupancy (proportion of occupied patches) compared to scale-free systems in both experimental and simulated systems. Under high dispersal conditions, we did not detect differences between types of metapopulations. Increasing patch degree (i.e. number of connections per patch) reduced the probability of extinction of local populations in both types of networks. We suggest the interaction between colonization/extinction rates and network topology alters the likelihood of rescue effects which results in differential patterns of occupancy and abundance in metapopulations.

Evolution alters post-invasion temporal dynamics in experimental communities.

The causes and consequences of temporal variation in the abundance of organisms constitute central themes in ecological inquiry. Rapid evolution can occur over ecological time-scales, potentially resulting in altered temporal variation in abundance and complicating inferences about the consequences of temporal variation. We assessed whether evolution altered the temporal variability in species' abundances in simple assemblages of species. We then compared experimental results to predictions from two-species models to better understand our results in the context of competitive and predator-prey interactions. We compared founder populations and their evolved descendants in experimental communities of ciliates and rotifers. Using a series of orthogonal contrasts, we then evaluated whether: (a) evolutionary history of invaders or (b) residents, (c) co-evolution among invaders and residents, and (d) invasion itself altered temporal variability in species abundances following invasion by a novel species. Using two-species competition and predator-prey models, we also generated predictions to better understand the effects of evolution on temporal variation in the abundances of interacting species. Finally, we compared experimental and modelling results to aid in the interpretation of which interspecific interactions might be affected by ongoing evolution in our communities. In experimental populations, differing evolutionary histories resulted in significant differences among treatments in abundances and temporal variation in abundances of both resident and invading species. For the contrasts, we found evidence that evolutionary history of the invader and residents, co-evolution among invaders and residents, and invasion itself affected temporal variability in abundance, but the importance of each differed for the two communities and the species within those communities. When comparing experimental results to model predictions, the increased abundance and decreased temporal variation in one invader, Euplotes daidaleos, are potentially consistent with evolution resulting in reduced attack rates in the novel community. Evolutionary history alone can affect temporal variation in the abundances of species, generating important consequences for interspecific interactions among species and complicating inferences about the consequences of temporal variability in biological communities.

Pervasive interactions between foliar microbes and soil nutrients mediate leaf production and herbivore damage in a tropical forest.

Producing and retaining leaves underlie the performance and survivorship of seedlings in deeply shaded tropical forests. These habitats are characterized by conditions ideal for foliar bacteria, which can be potent plant pathogens. Leaf production, retention and susceptibility to enemies may ultimately depend upon interactions among soil nutrients and foliar microbes, yet this has never been tested. We experimentally evaluated the degree that foliar bacteria and soil resource supply mediate leaf dynamics for five common tree species (five different families) in a Panamanian forest. We reduced foliar bacteria with antibiotics for 29 months and measured leaf production, retention and damage for seedlings nested within a replicated 15-yr factorial nutrient enrichment experiment (nitrogen, N; phosphorus, P; potassium, K). Our results demonstrate that when we applied antibiotics, soil nutrients - particularly N - always regulated seedling leaf production (and to a lesser extent herbivore damage) for all five tree species. In addition, it was common for two macronutrients together to negate or completely reverse the impact of applying either one alone. Our findings of frequent plant-microbe-nutrient interactions are novel and suggest that these interactions may reinforce plant species-environment associations, thereby creating a fairly cryptic and fine-scale dimension of niche differentiation for coexisting tree species.

Influences of Host Community Characteristics on Borrelia burgdorferi Infection Prevalence in Blacklegged Ticks.

Lyme disease is a major vector-borne bacterial disease in the USA. The disease is caused by Borrelia burgdorferi, and transmitted among hosts and humans, primarily by blacklegged ticks (Ixodes scapularis). The ~25 B. burgdorferi genotypes, based on genotypic variation of their outer surface protein C (ospC), can be phenotypically separated as strains that primarily cause human diseases-human invasive strains (HIS)-or those that rarely do. Additionally, the genotypes are non-randomly associated with host species. The goal of this study was to examine the extent to which phenotypic outcomes of B. burgdorferi could be explained by the host communities fed upon by blacklegged ticks. In 2006 and 2009, we determined the host community composition based on abundance estimates of the vertebrate hosts, and collected host-seeking nymphal ticks in 2007 and 2010 to determine the ospC genotypes within infected ticks. We regressed instances of B. burgdorferi phenotypes on site-specific characteristics of host communities by constructing Bayesian hierarchical models that properly handled missing data. The models provided quantitative support for the relevance of host composition on Lyme disease risk pertaining to B. burgdorferi prevalence (i.e. overall nymphal infection prevalence, or NIPAll) and HIS prevalence among the infected ticks (NIPHIS). In each year, NIPAll and NIPHIS was found to be associated with host relative abundances and diversity. For mice and chipmunks, the association with NIPAll was positive, but tended to be negative with NIPHIS in both years. However, the direction of association between shrew relative abundance with NIPAll or NIPHIS differed across the two years. And, diversity (H') had a negative association with NIPAll, but positive association with NIPHIS in both years. Our analyses highlight that the relationships between the relative abundances of three primary hosts and the community diversity with NIPAll, and NIPHIS, are variable in time and space, and that disease risk inference, based on the role of host community, changes when we examine risk overall or at the phenotypic level. Our discussion focuses on the observed relationships between prevalence and host community characteristics and how they substantiate the ecological understanding of phenotypic Lyme disease risk.

Foliar bacteria and soil fertility mediate seedling performance: a new and cryptic dimension of niche differentiation.

The phyllosphere (comprising the leaf surface and interior) is one of the world's largest microbial habitats and is host to an abundant and diverse array of bacteria. Nonetheless, the degree to which bacterial communities are benign, harmful, or beneficial to plants in situ is unknown. We tested the hypothesis that the net effect of reducing bacterial abundance and diversity would vary substantially among host species (from harmful to beneficial) and this would be strongly mediated by soil resource availability. To test this, we monitored tree seedling growth responses to commercial antibiotics among replicated resource supply treatments (N, P, K) in a tropical forest in Panama for 29 months. We applied either antibiotics or control water to replicated seedlings of five common tree species (Alseis blackiana, Desmopsis panamensis, Heisteria concinna, Sorocea affinis, and Tetragastris panamensis). These antibiotic treatments significantly reduced both the abundance and diversity of bacteria epiphytically as well as endophytically. Overall, the effect of antibiotics on performance was highly host specific. Applying antibiotics increased growth for three species by as much as 49% (Alseis, Heisteria, and Tetragastris), decreased growth for a fourth species by nearly 20% (Sorocea), and had no impact on a fifth species (Desmopsis). Perhaps more importantly, the degree to which foliar bacteria were harmful or not varied with soil resource supply. Specifically, applying antibiotics had no effect when potassium was added but increased growth rate by almost 40% in the absence of potassium. Alternatively, phosphorus enrichment caused the effect of bacteria to switch from being primarily beneficial to harmful or vice versa, but this depended entirely on the presence or absence of nitrogen enrichment (i.e., important and significant interactions). Our results are the first to demonstrate that the net effect of reducing the abundance and diversity of bacteria can have very strong positive and negative effects on seedling performance. Moreover, these effects were clearly mediated by soil resource availability. Though speculative, we suggest that foliar bacteria may interact with soil fertility to comprise an important, yet cryptic dimension of niche differentiation, which can have important implications for species coexistence.

Three Dimensional Human Neuro-Spheroid Model of Alzheimer's Disease Based on Differentiated Induced Pluripotent Stem Cells.

The testing of candidate drugs to slow progression of Alzheimer's disease (AD) requires clinical trials that are lengthy and expensive. Efforts to model the biochemical milieu of the AD brain may be greatly facilitated by combining two cutting edge technologies to generate three-dimensional (3D) human neuro-spheroid from induced pluripotent stem cells (iPSC) derived from AD subjects. We created iPSC from blood cells of five AD patients and differentiated them into 3D human neuronal culture. We characterized neuronal markers of our 3D neurons by immunocytochemical staining to validate the differentiation status. To block the generation of pathologic amyloid ß peptides (Aß), the 3D-differentiated AD neurons were treated with inhibitors targeting ß-secretase (BACE1) and γ-secretases. As predicted, both BACE1 and γ-secretase inhibitors dramatically decreased Aß generation in iPSC-derived neural cells derived from all five AD patients, under standard two-dimensional (2D) differentiation conditions. However, BACE1 and γ-secretase inhibitors showed less potency in decreasing Aß levels in neural cells differentiated under 3D culture conditions. Interestingly, in a single subject AD1, we found that BACE1 inhibitor treatment was not able to significantly reduce Aß42 levels. To investigate underlying molecular mechanisms, we performed proteomic analysis of 3D AD human neuronal cultures including AD1. Proteomic analysis revealed specific reduction of several proteins that might contribute to a poor inhibition of BACE1 in subject AD1. To our knowledge, this is the first iPSC-differentiated 3D neuro-spheroid model derived from AD patients' blood. Our results demonstrate that our 3D human neuro-spheroid model can be a physiologically relevant and valid model for testing efficacy of AD drug.

Evolution alters the consequences of invasions in experimental communities.

Evolution has the capacity to alter the course of biological invasions, although such changes remain mostly unexplored by experiments. Integrating evolution into studies of invasions is important, because species traits can potentially evolve in ways that either moderate or exacerbate the impacts of invasions. We have assessed whether species evolved during experimental invasions by comparing the performance of founder populations and their potentially evolved descendants in communities of ciliates and rotifers. Residents (analogous to native species) that have previous experience with invaders consistently reduced the performance of naive invaders, supporting the emergence of increased biotic resistance as one consequence of evolution during invasions. Experienced invaders exhibited both increased and decreased performance depending on the invader species considered. Through its influence on performance and species abundance, evolution also changed community composition during the course of invasions. The idiosyncratic patterns of evolutionary changes in invading and resident species complicate predictions about the long-term consequences of invasions from initial post-invasion dynamics.

Cell proliferation and neurogenesis in adult mouse brain.

Neurogenesis, the formation of new neurons, can be observed in the adult brain of many mammalian species, including humans. Despite significant progress in our understanding of adult neurogenesis, we are still missing data about the extent and location of production of neural precursors in the adult mammalian brain. We used 5-ethynyl-2'-deoxyuridine (EdU) to map the location of proliferating cells throughout the entire adult mouse brain and found that neurogenesis occurs at two locations in the mouse brain. The larger one we define as the main proliferative zone (MPZ), and the smaller one corresponds to the subgranular zone of the hippocampus. The MPZ can be divided into three parts. The caudate migratory stream (CMS) occupies the middle part of the MPZ. The cable of proliferating cells emanating from the most anterior part of the CMS toward the olfactory bulbs forms the rostral migratory stream. The thin layer of proliferating cells extending posteriorly from the CMS forms the midlayer. We have not found any additional aggregations of proliferating cells in the adult mouse brain that could suggest the existence of other major neurogenic zones in the adult mouse brain.

Occurrence and transmission efficiencies of Borrelia burgdorferi ospC types in avian and mammalian wildlife.

Borrelia burgdorferi s.s., the bacterium that causes Lyme disease in North America, circulates among a suite of vertebrate hosts and their tick vector. The bacterium can be differentiated at the outer surface protein C (ospC) locus into 25 genotypes. Wildlife hosts can be infected with a suite of ospC types but knowledge on the transmission efficiencies of these naturally infected hosts to ticks is still lacking. To evaluate the occupancy and detection of ospC types in wildlife hosts, we adapted a likelihood-based species patch occupancy model to test for the occurrence probabilities (ψ - "occupancy") and transmission efficiencies (ε - "detection") of each ospC type. We detected differences in ospC occurrence and transmission efficiencies from the null models with HIS (human invasive strains) types A and K having the highest occurrence estimates, but both HIS and non-HIS types having high transmission efficiencies. We also examined ospC frequency patterns with respect to strains known to be invasive in humans across the host species and phylogenetic groups. We found that shrews and to a lesser extent, birds, were important host groups supporting relatively greater frequencies of HIS to non-HIS types. This novel method of simultaneously assessing occurrence and transmission of ospC types provides a powerful tool in assessing disease risk at the genotypic level in naturally infected wildlife hosts and offers the opportunity to examine disease risk at the community level.

Home-Based Video Telehealth for Veterans With Dementia.

In-home video telehealth supplements office visits and offers comfort and convenience to patients with dementia and their caregivers.

Pyroglutamate-Aß 3 and 11 colocalize in amyloid plaques in Alzheimer's disease cerebral cortex with pyroglutamate-Aß 11 forming the central core.

N-terminal truncated amyloid beta (Aß) derivatives, especially the forms having pyroglutamate at the 3 position (AßpE3) or at the 11 position (AßpE11) have become the topic of considerable study. AßpE3 is known to make up a substantial portion of the Aß species in senile plaques while AßpE11 has received less attention. We have generated very specific polyclonal antibodies against both species. Each antibody recognizes only the antigen against which it was generated on Western blots and neither recognizes full length Aß. Both anti-AßpE3 and anti-AßpE11 stain senile plaques specifically in Alzheimer's disease cerebral cortex and colocalize with Aß, as shown by confocal microscopy. In a majority of plaques examined, AßpE11 was observed to be the dominant form in the innermost core. These data suggest that AßpE11 may serve as a generating site for senile plaque formation.

Retromer disruption promotes amyloidogenic APP processing.

Retromer deficiency has been implicated in sporadic AD and animals deficient in retromer components exhibit pronounced neurodegeneration. Because retromer performs retrograde transport from the endosome to the Golgi apparatus and neuronal Aß is found in late endosomal compartments, we speculated that retromer malfunction might enhance amyloidogenic APP processing by promoting interactions between APP and secretase enzymes in late endosomes. We have evaluated changes in amyloid precursor protein (APP) processing and trafficking as a result of disrupted retromer activity by knockdown of Vps35, a vacuolar sorting protein that is an essential component of the retromer complex. Knocking down retromer activity produced no change in the quantity or cellular distribution of total cellular APP and had no affect on internalization of cell-surface APP. Retromer deficiency did, however, increase the ratio of secreted Aß42:Aß40 in HEK-293 cells over-expressing APP695, due primarily to a decrease in Aß40 secretion. Recent studies suggest that the retromer-trafficked protein, Wntless, is secreted at the synapse in exosome vesicles and that these same vesicles contain Aß. We therefore hypothesized that retromer deficiency may be associated with altered exosomal secretion of APP and/or secretase fragments. Holo-APP, Presenilin and APP C-terminal fragments were detected in exosomal vesicles secreted from HEK-293 cells. Levels of total APP C-terminal fragments were significantly increased in exosomes secreted by retromer deficient cells. These data suggest that reduced retromer activity can mimic the effects of familial AD Presenilin mutations on APP processing and promote export of amyloidogenic APP derivatives.

Temperature fluctuation facilitates coexistence of competing species in experimental microbial communities.

1. Temperature fluctuation is a general phenomenon affecting many, if not all, species in nature. While a few studies have shown that temperature fluctuation can promote species coexistence, little is known about the effects of different regimes of temperature fluctuation on coexistence. 2. We experimentally investigated how temperature fluctuation and different regimes of temperature fluctuation ('red' environments in which temperature series exhibited positive temporal autocorrelation vs. 'white' environments in which temperature series showed little autocorrelation) affected the coexistence of two ciliated protists, Colpidium striatum Stein and Paramecium tetraurelia Sonneborn, which competed for bacterial resources. 3. We have previously shown that the two species differed in their growth responses to changes in temperature and in their resource utilization patterns. The two species were not always able to coexist at constant temperatures (22, 24, 26, 28 and 30 degrees C), with Paramecium being competitively excluded at 26 and 28 degrees C. This indicated that resource partitioning was insufficient to maintain coexistence at these temperatures. 4. Here we show that in both red and white environments in which temperature varied between 22 and 32 degrees C, Paramecium coexisted with Colpidium. Consistent with the differential effects of temperature on their intrinsic growth rates, Paramecium population dynamics were largely unaffected by temperature regimes, and Colpidium showed more variable population dynamics in the red environments. 5. Temperature-dependent competitive effects of Colpidium on Paramecium, together with resource partitioning, appeared to be responsible for the coexistence in the white environments; resource partitioning and the storage effect appeared to account for the coexistence in the red environments. 6. These results suggest that temperature fluctuation may play important roles in regulating species coexistence and diversity in ecological communities.

The vacuolar-ATPase inhibitor bafilomycin and mutant VPS35 inhibit canonical Wnt signaling.

Endosomal acidification and transport are essential functions in signal transduction. Recent data suggest that Wnt signaling requires intact endosomal transport machinery but the effects of endosomal acidification on Wnt signal transduction have not been evaluated. Here we report that bafilomycin, a specific inhibitor of the vacuolar proton ATPase that blocks endosomal acidification, inhibits canonical Wnt signal transduction initiated by Wnt ligand and partially inhibits signaling initiated by disheveled. Bafilomycin does not affect Tcf promoter activation by beta-catenin. These data indicate that endosomal acidification is necessary for Wnt signaling. To identify interactions between endosomal transport proteins and Wnt receptors, we performed a GST fusion protein pulldown experiment and identified a possible indirect interaction between the LRP6 intracellular domain and vacuolar protein sorting protein 35 (VPS35). We show that an N-terminal deletion mutant of VPS35 reduces canonical Wnt signaling in HEK-293 cells expressing exogenous Wnt-1. These data suggest that endosomal V-type ATPase activity and retromer trafficking proteins are functionally important in Wnt signal transduction.

Species richness and allometric scaling jointly determine biomass in model aquatic food webs.

1. Allometric theory makes specific predictions about how density, and consequently biomass, scale with organism size within trophic levels, across trophic levels and across food webs. 2. Diversity-yield relationships suggest that more diverse food webs can sometimes support more biomass through mechanisms involving niche complementarity or selection effects that are sometimes attributed to organism size. 3. We combine the above two approaches and show that, generally, density and biomass scale with organism size within and between trophic levels as predicted by allometric theory. Further, food webs converged in total biomass despite persistent differences in the composition and size of the organisms among food webs; species richness explained deviations from the constant yield of biomass expected from size-abundance relationships. 4. Our results suggest that organism size plays only a transient role in controlling community biomass because population increases or decreases lead to rapid convergence in biomass. Species richness affects community biomass independently by effectively increasing the mass of organisms that can be supported in a given productivity regime.

Population and community resilience in multitrophic communities.

Diversity-stability relationships have long been a topic of controversy in ecology, but one whose importance has been re-highlighted by increasing large-scale threats to global biodiversity. The ability of a community to recover from a perturbation (or resilience) is a common measure of stability that has received a large amount of theoretical attention. Yet, general expectations regarding diversity-resilience relations remain elusive. Moreover, the effects of productivity and its interaction with diversity on resilience are equally unclear. We examined the effects of species diversity, species composition, and productivity on population-and community-level resilience in experimental aquatic food webs composed of bacteria, algae, heterotrophic protozoa, and rotifers. Productivity manipulations were crossed with manipulations of the number of species and species compositions within trophic groups. Resilience was measured by perturbing communities with a nonselective, density-independent, mortality event and comparing responses over time between perturbed communities and controls. We found evidence that species diversity can enhance resilience at the community level (i.e., total community biomass), though this effect was more strongly expressed in low-productivity treatments. Diversity effects on resilience were driven by a sampling/selection effect, with resilient communities showing rapid response and dominance by a minority of species (primarily unicellular algae). In contrast, diversity had no effect on mean population-level resilience. Instead, the ability of a community's populations to recover from perturbations was dependent on species composition. We found no evidence of an effect of productivity, either positive or negative, on community- or population-level resilience. Our results indicate that the role of diversity as an insurer of stability may depend on the level of biological organization at which stability is measured, with effects emerging only when focusing on aggregate community properties.