Eyes of the world on a warmer, less frozen, and greener Arctic.

Rapid atmospheric warming and sea-ice retreat are driving widespread changes in Arctic ecosystems, among the most pervasive of which is the "greening of the Arctic"-an increase in the cover and biomass of vegetation observed by satellites across much of the Arctic tundra biome. Determining the drivers, impacts, and feedbacks of Arctic greening requires continued investment in robust field, remote-sensing, and model-based capabilities, and improved integration of the knowledge base of Arctic peoples. These tools and approaches support the triangulation of complex problems and the development of improved projections for the warmer Arctic tundra biome of the future.

The Chelate Effect Rationalizes Observed Rate Acceleration and Enantioselectivity in BINOL-Catalyzed Petasis Reactions.

Density functional theory (DFT) calculations afforded insight into the origin of the experimentally observed reaction rate acceleration (≥500 fold) and enantioselectivity (≥99 % ee) of 1,1'-bi-2-naphthol- (BINOL-) catalyzed three-component Petasis reactions . BINOL accelerates the rate determining step by forming a BIV chelate, which involves the loss of water from the hemiaminal moiety to generate an iminium intermediate. Subsequent vinyl group transfer from BIV to the iminium carbon affords the enantiomerically enriched product and a cyclic trigonal B(III)BINOL complex, which rapidly releases the BINOL allowing it to re-enter the catalytic cycle. In the transition state of the vinyl transfer step, C-H-O hydrogen bonding between the iminium C-H and O of (R)-BINOL directs the vinyl group addition to the Re-face of the iminium carbon. This mechanism explains both the rate acceleration and high enantioselectivity of the stereo determining step.

Bacterial Skin Assemblages of Sympatric Salamanders Are Primarily Shaped by Host Genus.

Bacterial assemblages on the skins of amphibians are known to influence pathogen resistance and other important physiological functions in the host. Host-specific factors and the environment play significant roles in structuring skin assemblages. This study used high-throughput 16S rRNA sequencing and multivariate analyses to examine differences in skin-bacterial assemblages from 246 salamanders belonging to three genera in the lungless family Plethodontidae along multiple spatial gradients. Composition and α- and ß-diversity of bacterial assemblages were defined, indicator species were identified for each host group, and the relative influences of host- versus environment-specific ecological factors were evaluated. At the broadest spatial scale, host genus, host species, and sampling site were predictive of skin assemblage structure, but host genus and species were more influential after controlling for the marginal effects of site, as well as nestedness of site. Furthermore, assemblage similarity within each host genus did not change with increasing geographic distance. At the smallest spatial scale, site-specific climate analyses revealed different relationships to climatic variables for each of the three genera, and these relationships were determined by host ecomode. Variation in bacterial assemblages of terrestrial hosts correlated with landscape-level climatic variability, and this pattern decayed with increasing water dependence of the host. Results from this study highlight host-specific considerations for researchers studying wildlife diseases in co-occurring, yet ecologically divergent, species.

Functional Redundancy in Bat Microbial Assemblage in the Presence of the White Nose Pathogen.

Understanding how host-associated microbial assemblages respond to pathogen invasion has implications for host health. Until recently, most investigations have focused on understanding the taxonomic composition of these assemblages. However, recent studies have suggested that microbial assemblage taxonomic composition is decoupled from its function, with assemblages being taxonomically varied but functionally constrained. The objective of this investigation was to understand how the Tri-colored bat, Perimyotis subflavus cutaneous microbial assemblage responds to fungal pathogen invasion within a functional context. We hypothesized that at a broad scale (e.g., KEGG pathways), there will be no difference in the functional assemblages between the white nose pathogen, Pseudogymnoascus destructans, positive and negative bats; and this pattern will be driven by the functional redundancy of bacterial taxa. At finer scales (e.g., gene models), we postulate differences in function attributed to interactions between bacteria and P. destructans, resulting in the production of antifungal metabolites. To test this, we used a combination of shotgun metagenomic and amplicon sequencing to characterize the bat cutaneous microbial assemblage in the presence/absence of P. destructans. Results showed that while there was a shift in taxonomic assemblage composition between P. destructans positive and negative bats, there was little overall difference in microbial function. Functional redundancy across bacterial taxa was clear at a broad-scale; however, both redundancy and variation in bacterial capability related to defense against pathogens was evident at finer scales. While functionality of the microbial assemblage was largely conserved in relation to P. destructans, the roles of particular functional pathways in resistance to fungal pathogens require further attention.

Testing the febrile response of snakes inoculated with Ophidiomyces ophidiicola, the causative agent of snake fungal disease.

Snake Fungal Disease (SFD) negatively impacts wild snake populations in the eastern United States and Europe. Ophidiomyces ophidiicola causes SFD and manifests clinically by the formation of heterophilic granulomas around the mouth and eyes, weight loss, impaired vision, and sometimes death. Field observations have documented early seasonal basking behaviors in severely infected snakes, potentially suggesting induction of a behavioral febrile response to combat the mycosis. This study tested the hypothesis that snakes inoculated with Ophidiomyces ophidiicola would seek elevated basking temperatures to control body temperature and behaviorally induce a febrile response. Eastern ribbon snakes (Thamnophis saurita, n = 29) were experimentally or sham inoculated with O. ophidiicola. Seven days after inoculation, snakes were tested on a thermal gradient and the internal body temperature and substrate temperature of each snake was recorded over time. Quantitative PCR was used when snakes arrived, during pre-inoculation, and post-inoculation to test snakes for the presence of O. ophidiicola. Some snakes arrived with O. ophidiicola and were subsequently inoculated, allowing for an assessment of secondary exposure effects. Snake thermoregulatory behavior was compared between 1) O. ophidiicola inoculated vs. sham inoculated treatments, 2) infected vs. disease negative groups, and 3) disease naïve vs. pre-exposed immune response categories. Neither internal nor substrate temperatures differed among initially prescribed, and qPCR recovered disease states, although infected snakes tended to reach a preferred body temperature faster than disease negative snakes. Snakes experiencing their first exposure (disease naïve) sought higher substrate temperatures than snakes experiencing their second exposure (pre-exposed). Here, we recover no evidence for behaviorally induced fever in snakes with SFD but do elucidate a febrile immune response associated with secondary exposure.

Distinct microbial assemblages associated with genetic selection for high- and low- muscle yield in rainbow trout.

BACKGROUND: Fish gut microbial assemblages play a crucial role in the growth rate, metabolism, and immunity of the host. We hypothesized that the gut microbiota of rainbow trout was correlated with breeding program based genetic selection for muscle yield. To test this hypothesis, fecal samples from 19 fish representing an F2 high-muscle genetic line (ARS-FY-H) and 20 fish representing an F1 low-muscle yield genetic line (ARS-FY-L) were chosen for microbiota profiling using the 16S rRNA gene. Significant differences in microbial assemblages between these two genetic lines might represent the effect of host genetic selection in structuring the gut microbiota of the host. RESULTS: Tukey's transformed inverse Simpson indices indicated that high muscle yield genetic line (ARS-FY-H) samples have higher microbial diversity compared to those of the low muscle yield genetic line (ARS-FY-L) (LMM, χ2(1) =14.11, p < 0.05). The fecal samples showed statistically distinct structure in microbial assemblages between the genetic lines (F1,36 = 4.7, p < 0.05, R2 = 11.9%). Functional profiling of bacterial operational taxonomic units predicted characteristic functional capabilities of the microbial communities in the high (ARS-FY-H) and low (ARS-FY-L) muscle yield genetic line samples. CONCLUSION: The significant differences of the microbial assemblages between high (ARS-FY-H) and low (ARS-FY-L) muscle yield genetic lines indicate a possible effect of genetic selection on the microbial diversity of the host. The functional composition of taxa demonstrates a correlation between bacteria and improving the muscle accretion in the host, probably, by producing various metabolites and enzymes that might aid in digestion. Further research is required to elucidate the mechanisms involved in shaping the microbial community through host genetic selection.

Fast, Robust, and Sensitive Identification of Residual Host Cell Proteins in Recombinant Monoclonal Antibodies Using Sodium Deoxycholate Assisted Digestion.

Residual host cell proteins (HCPs) present in biotherapeutics can pose potential safety risks for patients or affect product stability, thus prompting a critical need to monitor HCPs in drug substance or product to ensure product safety and quality. Current approaches for robust HCP identification at or above 10 ppm levels require either concatenated peptide fractionation or enrichment via antibody depletion, which challenges the direct quantitation of HCPs. This paper describes a simple, fast sample preparation method without the need for sample fractionation or enrichment; instead, we utilize trypsin-friendly sodium deoxycholate (SDC) as an advantageous denaturant that can be effectively removed following acidification at the end of sample digestion. This new approach enables the end-to-end one-dimensional liquid chromatography-tandem mass spectrometry (1D LC-MS/MS) workflow (i.e., from sample preparation to HCP identification) to be completed in 7-8 h while demonstrating the ability to consistently identify HCPs across a broad molecular weight range at 10 ppm or above.

Landscape impacts of 3D-seismic surveys in the Arctic National Wildlife Refuge, Alaska.

Although three-dimensional (3D) seismic surveys have improved the success rate of exploratory drilling for oil and gas, the impacts have received little scientific scrutiny, despite affecting more area than any other oil and gas activity. To aid policy-makers and scientists, we reviewed studies of the landscape impacts of 3D-seismic surveys in the Arctic. We analyzed a proposed 3D-seismic program in northeast Alaska, in the northern Arctic National Wildlife Refuge, which includes a grid 63,000 km of seismic trails and additional camp-move trails. Current regulations are not adequate to eliminate impacts from these activities. We address issues related to the high-density of 3D trails compared to 2D methods, with larger crews, more camps, and more vehicles. We focus on consequences to the hilly landscapes, including microtopography, snow, vegetation, hydrology, active layers, and permafrost. Based on studies of 2D-seismic trails created in 1984-1985 in the same area by similar types of vehicles, under similar regulations, approximately 122 km2 would likely sustain direct medium- to high-level disturbance from the proposed exploration, with possibly expanded impacts through permafrost degradation and hydrological connectivity. Strong winds are common, and snow cover necessary to minimize impacts from vehicles is windblown and inadequate to protect much of the area. Studies of 2D-seismic impacts have shown that moist vegetation types, which dominate the area, sustain longer-lasting damage than wet or dry types, and that the heavy vehicles used for mobile camps caused the most damage. The permafrost is ice rich, which combined with the hilly topography, makes it especially susceptible to thermokarst and erosion triggered by winter vehicle traffic. The effects of climate warming will exacerbate the impacts of winter travel due to warmer permafrost and a shift of precipitation from snow to rain. The cumulative impacts of 3D-seismic traffic in tundra areas need to be better assessed, together with the effects of climate change and the industrial development that would likely follow. Current data needs include studies of the impacts of 3D-seismic exploration, better climate records for the Arctic National Wildlife Refuge, especially for wind and snow; and high-resolution maps of topography, ground ice, hydrology, and vegetation.

Microbial Assemblage Dynamics Within the American Alligator Nesting Ecosystem: a Comparative Approach Across Ecological Scales.

Understanding the ecological processes that shape species assemblage patterns is central to community ecology. The effects of ecological processes on assemblage patterns are scale-dependent. We used metabarcoding and shotgun sequencing to determine bacterial taxonomic and functional assemblage patterns among varying defined focal scales (micro-, meso-, and macroscale) within the American alligator (Alligator mississippiensis) nesting microbiome. We correlate bacterial assemblage patterns among eight nesting compartments within and proximal to alligator nests (micro-), across 18 nests (meso-), and between 4 geographic sampling sites (macro-), to determine which ecological processes may drive bacterial assemblage patterns within the nesting environment. Among all focal scales, bacterial taxonomic and functional richness (α-diversity) did not statistically differ. In contrast, bacterial assemblage structure (ß-diversity) was unique across all focal scales, whereas functional pathways were redundant within nests and across geographic sites. Considering these observed scale-based patterns, taxonomic bacterial composition may be governed by unique environmental filters and dispersal limitations relative to microbial functional attributes within the alligator nesting environment. These results advance pattern-process dynamics within the field of microbial community ecology and describe processes influencing the American alligator nest microbiome.

Variation in the Slimy Salamander (Plethodon spp.) Skin and Gut-Microbial Assemblages Is Explained by Geographic Distance and Host Affinity.

A multicellular host and its microbial communities are recognized as a metaorganism-a composite unit of evolution. Microbial communities have a variety of positive and negative effects on the host life history, ecology, and evolution. This study used high-throughput amplicon sequencing to characterize the complete skin and gut microbial communities, including both bacteria and fungi, of a terrestrial salamander, Plethodon glutinosus (Family Plethodontidae). We assessed salamander populations, representing nine mitochondrial haplotypes ('clades'), for differences in microbial assemblages across 13 geographic locations in the Southeastern United States. We hypothesized that microbial assemblages were structured by both host factors and geographic distance. We found a strong correlation between all microbial assemblages at close geographic distances, whereas, as spatial distance increases, the patterns became increasingly discriminate. Network analyses revealed that gut-bacterial communities have the highest degree of connectedness across geographic space. Host salamander clade was explanatory of skin-bacterial and gut-fungal assemblages but not gut-bacterial assemblages, unless the latter were analyzed within a phylogenetic context. We also inferred the function of gut-fungal assemblages to understand how an understudied component of the gut microbiome may influence salamander life history. We concluded that dispersal limitation may in part describe patterns in microbial assemblages across space and also that the salamander host may select for skin and gut communities that are maintained over time in closely related salamander populations.

Analysis of the fecal microbiota of fast- and slow-growing rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Diverse microbial communities colonizing the intestine of fish contribute to their growth, digestion, nutrition, and immune function. We hypothesized that fecal samples representing the gut microbiota of rainbow trout could be associated with differential growth rates observed in fish breeding programs. If true, harnessing the functionality of this microbiota can improve the profitability of aquaculture. The first objective of this study was to test this hypothesis if gut microbiota is associated with fish growth rate (body weight). Four full-sibling families were stocked in the same tank and fed an identical diet. Two fast-growing and two slow-growing fish were selected from each family for 16S rRNA microbiota profiling. Microbiota diversity varies with different DNA extraction methods. The second objective of this study was to compare the effects of five commonly used DNA extraction methods on the microbiota profiling and to determine the most appropriate extraction method for this study. These methods were Promega-Maxwell, Phenol-chloroform, MO-BIO, Qiagen-Blood/Tissue, and Qiagen-Stool. Methods were compared according to DNA integrity, cost, feasibility and inter-sample variation based on non-metric multidimensional scaling ordination (nMDS) clusters. RESULTS: Differences in DNA extraction methods resulted in significant variation in the identification of bacteria that compose the gut microbiota. Promega-Maxwell had the lowest inter-sample variation and was therefore used for the subsequent analyses. Beta diversity of the bacterial communities showed significant variation between breeding families but not between the fast- and slow-growing fish. However, an indicator analysis determined that cellulose, amylose degrading and amino acid fermenting bacteria (Clostridium, Leptotrichia, and Peptostreptococcus) are indicator taxa of the fast-growing fish. In contrary, pathogenic bacteria (Corynebacterium and Paeniclostridium) were identified as indicator taxa for the slow-growing fish. CONCLUSION: DNA extraction methodology should be carefully considered for accurate profiling of the gut microbiota. Although the microbiota was not significantly different between the fast- and slow-growing fish groups, some bacterial taxa with functional implications were indicative of fish growth rate. Further studies are warranted to explore how bacteria are transmitted and potential usage of the indicator bacteria of fast-growing fish for development of probiotics that may improve fish health and growth.

Changes in timing of seasonal peak photosynthetic activity in northern ecosystems.

Seasonality in photosynthetic activity is a critical component of seasonal carbon, water, and energy cycles in the Earth system. This characteristic is a consequence of plant's adaptive evolutionary processes to a given set of environmental conditions. Changing climate in northern lands (>30°N) alters the state of climatic constraints on plant growth, and therefore, changes in the seasonality and carbon accumulation are anticipated. However, how photosynthetic seasonality evolved to its current state, and what role climatic constraints and their variability played in this process and ultimately in carbon cycle is still poorly understood due to its complexity. Here, we take the "laws of minimum" as a basis and introduce a new framework where the timing (day of year) of peak photosynthetic activity (DOYPmax ) acts as a proxy for plant's adaptive state to climatic constraints on its growth. Our analyses confirm that spatial variations in DOYPmax reflect spatial gradients in climatic constraints as well as seasonal maximum and total productivity. We find a widespread warming-induced advance in DOYPmax (-1.66 ± 0.30 days/decade, p < 0.001) across northern lands, indicating a spatiotemporal dynamism of climatic constraints to plant growth. We show that the observed changes in DOYPmax are associated with an increase in total gross primary productivity through enhanced carbon assimilation early in the growing season, which leads to an earlier phase shift in land-atmosphere carbon fluxes and an increase in their amplitude. Such changes are expected to continue in the future based on our analysis of earth system model projections. Our study provides a simplified, yet realistic framework based on first principles for the complex mechanisms by which various climatic factors constrain plant growth in northern ecosystems.

Vegetation on mesic loamy and sandy soils along a 1700-km maritime Eurasia Arctic Transect.

QUESTIONS: How do plant communities on zonal loamy vs. sandy soils vary across the full maritime Arctic bioclimate gradient? How are plant communities of these areas related to existing vegetation units of the European Vegetation Classification? What are the main environmental factors controlling transitions of vegetation along the bioclimate gradient? LOCATION: 1700-km Eurasia Arctic Transect (EAT), Yamal Peninsula and Franz Josef Land (FJL), Russia. METHODS: The Braun-Blanquet approach was used to sample mesic loamy and sandy plots on 14 total study sites at six locations, one in each of the five Arctic bioclimate subzones and the forest-tundra transition. Trends in soil factors, cover of plant growth forms (PGFs) and species diversity were examined along the summer warmth index (SWI) gradient and on loamy and sandy soils. Classification and ordination were used to group the plots and to test relationships between vegetation and environmental factors. RESULTS: Clear, mostly non-linear, trends occurred for soil factors, vegetation structure and species diversity along the climate gradient. Cluster analysis revealed seven groups with clear relationships to subzone and soil texture. Clusters at the ends of the bioclimate gradient (forest-tundra and polar desert) had many highly diagnostic taxa, whereas clusters from the Yamal Peninsula had only a few. Axis 1 of a DCA was strongly correlated with latitude and summer warmth; Axis 2 was strongly correlated with soil moisture, percentage sand and landscape age. CONCLUSIONS: Summer temperature and soil texture have clear effects on tundra canopy structure and species composition, with consequences for ecosystem properties. Each layer of the plant canopy has a distinct region of peak abundance along the bioclimate gradient. The major vegetation types are weakly aligned with described classes of the European Vegetation Checklist, indicating a continuous floristic gradient rather than distinct subzone regions. The study provides ground-based vegetation data for satellite-based interpretations of the western maritime Eurasian Arctic, and the first vegetation data from Hayes Island, Franz Josef Land, which is strongly separated geographically and floristically from the rest of the gradient and most susceptible to on-going climate change.

A 2D LC-MS/MS Strategy for Reliable Detection of 10-ppm Level Residual Host Cell Proteins in Therapeutic Antibodies.

Methodologies employing LC-MS/MS have been increasingly used for characterization and identification of residual host cell proteins (HCPs) in biopharmaceutical products to ensure their consistent product quality and safety for patients. To improve the sensitivity and reliability for HCP detection, we developed a high pH-low pH two-dimensional reversed phase LC-MS/MS approach in conjunction with offline fraction concatenation. Proof-of -concept was established using a model in which seven proteins spanning a size range of 29-78 kDa are spiked into a purified antibody product to simulate the presence of low-level HCPs. By incorporating a tandem column configuration and a shallow gradient through the second-dimension, all seven proteins were consistently identified at 10 ppm with 100% success rate following LC-MS/MS analysis of six concatenated fractions across multiple analysts, column lots and injection loads. Using the more complex Universal Proteomic Standard 1 (UPS-1) as an HCP model, positive identification was consistently achieved for 19 of the 22 proteins in 8-12 ppm range (10 ppm ±20%). For the first time, we demonstrate an effective LC-MS/MS strategy that not only has high sensitivity but also high reliability for HCP detection. The method performance has high impact on pharmaceutical company practices in using advanced LC-MS/MS technology to ensure product quality and patient safety.

Which species, how many, and from where: Integrating habitat suitability, population genomics, and abundance estimates into species reintroduction planning.

Extirpated organisms are reintroduced into their former ranges worldwide to combat species declines and biodiversity losses. The growing field of reintroduction biology provides guiding principles for reestablishing populations, though criticisms remain regarding limited integration of initial planning, modeling frameworks, interdisciplinary collaborations, and multispecies approaches. We used an interdisciplinary, multispecies, quantitative framework to plan reintroductions of three fish species into Abrams Creek, Great Smoky Mountains National Park, USA. We first assessed the appropriateness of habitat at reintroduction sites for banded sculpin (Cottus carolinae), greenside darter (Etheostoma blennioides), and mottled sculpin (Cottus bairdii) using species distribution modeling. Next, we evaluated the relative suitability of nine potential source stock sites using population genomics, abundance estimates, and multiple-criteria decision analysis (MCDA) based on known correlates of reintroduction success. Species distribution modeling identified mottled sculpin as a poor candidate, but banded sculpin and greenside darter as suitable candidates for reintroduction based on species-habitat relationships and habitats available in Abrams Creek. Genotyping by sequencing revealed acceptable levels of genetic diversity at all candidate source stock sites, identified population clusters, and allowed for estimating the number of fish that should be included in translocations. Finally, MCDA highlighted priorities among candidate source stock sites that were most likely to yield successful reintroductions based on differential weightings of habitat assessment, population genomics, and the number of fish available for translocation. Our integrative approach represents a unification of multiple recent advancements in the field of reintroduction biology and highlights the benefit of shifting away from simply choosing nearby populations for translocation to an information-based science with strong a priori planning coupled with several suggested posteriori monitoring objectives. Our framework can be applied to optimize reintroduction successes for a multitude of organisms and advances in the science of reintroduction biology by simultaneously addressing a variety of past criticisms of the field.

Circumpolar arctic tundra biomass and productivity dynamics in response to projected climate change and herbivory.

Satellite remote sensing data have indicated a general 'greening' trend in the arctic tundra biome. However, the observed changes based on remote sensing are the result of multiple environmental drivers, and the effects of individual controls such as warming, herbivory, and other disturbances on changes in vegetation biomass, community structure, and ecosystem function remain unclear. We apply ArcVeg, an arctic tundra vegetation dynamics model, to estimate potential changes in vegetation biomass and net primary production (NPP) at the plant community and functional type levels. ArcVeg is driven by soil nitrogen output from the Terrestrial Ecosystem Model, existing densities of Rangifer populations, and projected summer temperature changes by the NCAR CCSM4.0 general circulation model across the Arctic. We quantified the changes in aboveground biomass and NPP resulting from (i) observed herbivory only; (ii) projected climate change only; and (iii) coupled effects of projected climate change and herbivory. We evaluated model outputs of the absolute and relative differences in biomass and NPP by country, bioclimate subzone, and floristic province. Estimated potential biomass increases resulting from temperature increase only are approximately 5% greater than the biomass modeled due to coupled warming and herbivory. Such potential increases are greater in areas currently occupied by large or dense Rangifer herds such as the Nenets-occupied regions in Russia (27% greater vegetation increase without herbivores). In addition, herbivory modulates shifts in plant community structure caused by warming. Plant functional types such as shrubs and mosses were affected to a greater degree than other functional types by either warming or herbivory or coupled effects of the two.

Sequence-based classification and identification of Fungi.

Fungal taxonomy and ecology have been revolutionized by the application of molecular methods and both have increasing connections to genomics and functional biology. However, data streams from traditional specimen- and culture-based systematics are not yet fully integrated with those from metagenomic and metatranscriptomic studies, which limits understanding of the taxonomic diversity and metabolic properties of fungal communities. This article reviews current resources, needs, and opportunities for sequence-based classification and identification (SBCI) in fungi as well as related efforts in prokaryotes. To realize the full potential of fungal SBCI it will be necessary to make advances in multiple areas. Improvements in sequencing methods, including long-read and single-cell technologies, will empower fungal molecular ecologists to look beyond ITS and current shotgun metagenomics approaches. Data quality and accessibility will be enhanced by attention to data and metadata standards and rigorous enforcement of policies for deposition of data and workflows. Taxonomic communities will need to develop best practices for molecular characterization in their focal clades, while also contributing to globally useful datasets including ITS. Changes to nomenclatural rules are needed to enable validPUBLICation of sequence-based taxon descriptions. Finally, cultural shifts are necessary to promote adoption of SBCI and to accord professional credit to individuals who contribute to community resources.

More similar than different: Host cell protein production using three null CHO cell lines.

To understand the diversity in the cell culture harvest (i.e., feedstock) provided for downstream processing, we compared host cell protein (HCP) profiles using three Chinese Hamster Ovary (CHO) cell lines in null runs which did not generate any recombinant product. Despite differences in CHO lineage, upstream process, and culture performance, the cell lines yielded similar cell-specific productivities for immunogenic HCPs. To compare the dynamics of HCP production, we searched for correlations between the time-course profiles of HCP (as measured by multi-analyte ELISA) and those of two intracellular HCP species, phospholipase B-like 2 (PLBL2) and lactate dehydrogenase (LDH). Across the cell lines, proteins in the day 14 supernatants analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) showed different spot patterns. However, subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) indicated otherwise: the total number of peptides and proteins identified were comparable, and 80% of the top 1,000 proteins identified were common to all three lines. Finally, to assess the impact of culture viability on extracellular HCP profiles, we analyzed supernatants from a cell line whose viability dropped after day 10. The amounts of HCP and PLBL2 (quantified by their respective ELISAs) as well as the numbers and major populations of HCPs (identified by LC-MS/MS) were similar across days 10, 14, and 17, during which viabilities declined from ∼80% to <20% and extracellular LDH levels increased several-fold. Our findings indicate that the CHO-derived HCPs in the feedstock for downstream processing may not be as diverse across cell lines and upstream processes, or change as dramatically upon viability decline as originally expected. In addition, our findings show that high density CHO cultures (>10(7) cells/mL)-operated in fed-batch mode and exhibiting high viabilities (>70%) throughout the culture duration-can accumulate a considerable amount of immunogenic HCP (∼1-2 g/L) in the extracellular environment at the time of harvest (day 14). This work also demonstrates the potential of using LC-MS/MS to overcome the limitations associated with ELISA and 2D-PAGE for HCP analysis.

Secreted amyloid ß-proteins in a cell culture model include N-terminally extended peptides that impair synaptic plasticity.

Evidence for a central role of amyloid ß-protein (Aß) in the genesis of Alzheimer's disease (AD) has led to advanced human trials of Aß-lowering agents. The "amyloid hypothesis" of AD postulates deleterious effects of small, soluble forms of Aß on synaptic form and function. Because selectively targeting synaptotoxic forms of soluble Aß could be therapeutically advantageous, it is important to understand the full range of soluble Aß derivatives. We previously described a Chinese hamster ovary (CHO) cell line (7PA2 cells) that stably expresses mutant human amyloid precursor protein (APP). Here, we extend this work by purifying an sodium dodecyl sulfate (SDS)-stable, ∼8 kDa Aß species from the 7PA2 medium. Mass spectrometry confirmed its identity as a noncovalently bonded Aß40 homodimer that impaired hippocampal long-term potentiation (LTP) in vivo. We further report the detection of Aß-containing fragments of APP in the 7PA2 medium that extend N-terminal from Asp1 of Aß. These N-terminally extended Aß-containing monomeric fragments are distinct from soluble Aß oligomers formed from Aß1-40/42 monomers and are bioactive synaptotoxins secreted by 7PA2 cells. Importantly, decreasing ß-secretase processing of APP elevated these alternative synaptotoxic APP fragments. We conclude that certain synaptotoxic Aß-containing species can arise from APP processing events N-terminal to the classical ß-secretase cleavage site.

Cumulative geoecological effects of 62 years of infrastructure and climate change in ice-rich permafrost landscapes, Prudhoe Bay Oilfield, Alaska.

Many areas of the Arctic are simultaneously affected by rapid climate change and rapid industrial development. These areas are likely to increase in number and size as sea ice melts and abundant Arctic natural resources become more accessible. Documenting the changes that have already occurred is essential to inform management approaches to minimize the impacts of future activities. Here, we determine the cumulative geoecological effects of 62 years (1949-2011) of infrastructure- and climate-related changes in the Prudhoe Bay Oilfield, the oldest and most extensive industrial complex in the Arctic, and an area with extensive ice-rich permafrost that is extraordinarily sensitive to climate change. We demonstrate that thermokarst has recently affected broad areas of the entire region, and that a sudden increase in the area affected began shortly after 1990 corresponding to a rapid rise in regional summer air temperatures and related permafrost temperatures. We also present a conceptual model that describes how infrastructure-related factors, including road dust and roadside flooding are contributing to more extensive thermokarst in areas adjacent to roads and gravel pads. We mapped the historical infrastructure changes for the Alaska North Slope oilfields for 10 dates from the initial oil discovery in 1968-2011. By 2010, over 34% of the intensively mapped area was affected by oil development. In addition, between 1990 and 2001, coincident with strong atmospheric warming during the 1990s, 19% of the remaining natural landscapes (excluding areas covered by infrastructure, lakes and river floodplains) exhibited expansion of thermokarst features resulting in more abundant small ponds, greater microrelief, more active lakeshore erosion and increased landscape and habitat heterogeneity. This transition to a new geoecological regime will have impacts to wildlife habitat, local residents and industry.