Snagfall the first decade after severe bark beetle infestation of high-elevation forests in Colorado, USA.

The persistence and fall rate of snags (standing dead trees) generated during bark beetle outbreaks have consequences for the behavior, effects, and suppression of potential wildfires, hazard tree and timber salvage operations, wildlife habitat, and numerous ecosystem processes. However, post-beetle snagfall dynamics are poorly understood in most forest types. We tagged standing live and dead lodgepole pine (Pinus contorta), subalpine fir (Abies lasiocarpa), and Engelmann spruce (Picea engelmannii), including beetle-killed pine snags following the peak of a recent mountain pine bark beetle outbreak in watersheds at the Fraser Experimental Forest in northcentral Colorado and sampled snagfall 10 and 12 years later. Bark beetle attacks began in 2003, peaked by 2006, and killed 78% of overstory lodgepole pine in 133 plots distributed across a range of stand and site conditions. Of those snags, only 17% fell between 2007 and 2018. Most snags broke at ground level, due to butt rot, and were oriented downhill. In contrast, snags that tipped up or snapped off above the ground were oriented with the prevailing winds. Equal numbers of snags fell singly and in multiple-tree groups, and equal numbers remained elevated rather than in contact with the ground. Lodgepole pine snagfall was 1.6-times higher on steep slopes (>40%) where dead pine density was higher, compared to flatter sites. Based on our findings and previous research, we estimate that one-half the beetle-killed lodgepole pine in high-elevation forests such as those at Fraser may fall within 15-20 yr of beetle infestation, but that some pine snags are likely to persist for decades longer. Post-outbreak snagfall dynamics create a multiple-decade legacy of bark beetle outbreaks that will persist longer in high-elevation compared to lower-elevation forests.

Changing forest water yields in response to climate warming: results from long-term experimental watershed sites across North America.

Climate warming is projected to affect forest water yields but the effects are expected to vary. We investigated how forest type and age affect water yield resilience to climate warming. To answer this question, we examined the variability in historical water yields at long-term experimental catchments across Canada and the United States over 5-year cool and warm periods. Using the theoretical framework of the Budyko curve, we calculated the effects of climate warming on the annual partitioning of precipitation (P) into evapotranspiration (ET) and water yield. Deviation (d) was defined as a catchment's change in actual ET divided by P [AET/P; evaporative index (EI)] coincident with a shift from a cool to a warm period - a positive d indicates an upward shift in EI and smaller than expected water yields, and a negative d indicates a downward shift in EI and larger than expected water yields. Elasticity was defined as the ratio of interannual variation in potential ET divided by P (PET/P; dryness index) to interannual variation in the EI - high elasticity indicates low d despite large range in drying index (i.e., resilient water yields), low elasticity indicates high d despite small range in drying index (i.e., nonresilient water yields). Although the data needed to fully evaluate ecosystems based on these metrics are limited, we were able to identify some characteristics of response among forest types. Alpine sites showed the greatest sensitivity to climate warming with any warming leading to increased water yields. Conifer forests included catchments with lowest elasticity and stable to larger water yields. Deciduous forests included catchments with intermediate elasticity and stable to smaller water yields. Mixed coniferous/deciduous forests included catchments with highest elasticity and stable water yields. Forest type appeared to influence the resilience of catchment water yields to climate warming, with conifer and deciduous catchments more susceptible to climate warming than the more diverse mixed forest catchments.

Persistent reduced ecosystem respiration after insect disturbance in high elevation forests.

Amid a worldwide increase in tree mortality, mountain pine beetles (Dendroctonus ponderosae Hopkins) have led to the death of billions of trees from Mexico to Alaska since 2000. This is predicted to have important carbon, water and energy balance feedbacks on the Earth system. Counter to current projections, we show that on a decadal scale, tree mortality causes no increase in ecosystem respiration from scales of several square metres up to an 84 km(2) valley. Rather, we found comparable declines in both gross primary productivity and respiration suggesting little change in net flux, with a transitory recovery of respiration 6-7 years after mortality associated with increased incorporation of leaf litter C into soil organic matter, followed by further decline in years 8-10. The mechanism of the impact of tree mortality caused by these biotic disturbances is consistent with reduced input rather than increased output of carbon.

Quantifying effects of snow depth on caribou winter range selection and movement in Arctic Alaska.

BACKGROUND: Caribou and reindeer across the Arctic spend more than two thirds of their lives moving in snow. Yet snow-specific mechanisms driving their winter ecology and potentially influencing herd health and movement patterns are not well known. Integrative research coupling snow and wildlife sciences using observations, models, and wildlife tracking technologies can help fill this knowledge void. METHODS: Here, we quantified the effects of snow depth on caribou winter range selection and movement. We used location data of Central Arctic Herd (CAH) caribou in Arctic Alaska collected from 2014 to 2020 and spatially distributed and temporally evolving snow depth data produced by SnowModel. These landscape-scale (90 m), daily snow depth data reproduced the observed spatial snow-depth variability across typical areal extents occupied by a wintering caribou during a 24-h period. RESULTS: We found that fall snow depths encountered by the herd north of the Brooks Range exerted a strong influence on selection of two distinct winter range locations. In winters with relatively shallow fall snow depth (2016/17, 2018/19, and 2019/20), the majority of the CAH wintered on the tundra north of the Brooks Range mountains. In contrast, during the winters with relatively deep fall snow depth (2014/15, 2015/16, and 2017/18), the majority of the CAH caribou wintered in the mountainous boreal forest south of the Brooks Range. Long-term (19 winters; 2001-2020) monitoring of CAH caribou winter distributions confirmed this relationship. Additionally, snow depth affected movement and selection differently within these two habitats: in the mountainous boreal forest, caribou avoided areas with deeper snow, but when on the tundra, snow depth did not trigger significant deep-snow avoidance. In both wintering habitats, CAH caribou selected areas with higher lichen abundance, and they moved significantly slower when encountering deeper snow. CONCLUSIONS: In general, our findings indicate that regional-scale selection of winter range is influenced by snow depth at or prior to fall migration. During winter, daily decision-making within the winter range is driven largely by snow depth. This integrative approach of coupling snow and wildlife observations with snow-evolution and caribou-movement modeling to quantify the multi-facetted effects of snow on wildlife ecology is applicable to caribou and reindeer herds throughout the Arctic.

A Lagrangian Snow-Evolution System for Sea-Ice Applications (SnowModel-LG): Part I-Model Description.

A Lagrangian snow-evolution model (SnowModel-LG) was used to produce daily, pan-Arctic, snow-on-sea-ice, snow property distributions on a 25 × 25-km grid, from 1 August 1980 through 31 July 2018 (38 years). The model was forced with NASA's Modern Era Retrospective-Analysis for Research and Applications-Version 2 (MERRA-2) and European Centre for Medium-Range Weather Forecasts (ECMWF) ReAnalysis-5th Generation (ERA5) atmospheric reanalyses, and National Snow and Ice Data Center (NSIDC) sea ice parcel concentration and trajectory data sets (approximately 61,000, 14 × 14-km parcels). The simulations performed full surface and internal energy and mass balances within a multilayer snowpack evolution system. Processes and features accounted for included rainfall, snowfall, sublimation from static-surfaces and blowing-snow, snow melt, snow density evolution, snow temperature profiles, energy and mass transfers within the snowpack, superimposed ice, and ice dynamics. The simulations produced horizontal snow spatial structures that likely exist in the natural system but have not been revealed in previous studies spanning these spatial and temporal domains. Blowing-snow sublimation made a significant contribution to the snowpack mass budget. The superimposed ice layer was minimal and decreased over the last four decades. Snow carryover to the next accumulation season was minimal and sensitive to the melt-season atmospheric forcing (e.g., the average summer melt period was 3 weeks or 50% longer with ERA5 forcing than MERRA-2 forcing). Observed ice dynamics controlled the ice parcel age (in days), and ice age exerted a first-order control on snow property evolution.

O antigen protects Bordetella parapertussis from complement.

Bordetella pertussis, a causative agent of whooping cough, expresses BrkA, which confers serum resistance, but the closely related human pathogen that also causes whooping cough, Bordetella parapertussis, does not. Interestingly, B. parapertussis, but not B. pertussis, produces an O antigen, a factor shown in other models to confer serum resistance. Using a murine model of infection, we determined that O antigen contributes to the ability of B. parapertussis to colonize the respiratory tract during the first week of infection, but not thereafter. Interestingly, an O antigen-deficient strain of B. parapertussis was not defective in colonizing mice lacking the complement cascade. O antigen prevented both complement component C3 deposition on the surface and complement-mediated killing of B. parapertussis. In addition, O antigen was required for B. parapertussis to systemically spread in complement-sufficient mice, but not complement-deficient mice. These data indicate that O antigen enables B. parapertussis to efficiently colonize the lower respiratory tract by protecting against complement-mediated control and clearance.

Modification of the Creator recombination system for proteomics applications--improved expression by addition of splice sites.

BACKGROUND: Recombinational systems have been developed to rapidly shuttle Open Reading Frames (ORFs) into multiple expression vectors in order to analyze the large number of cDNAs available in the post-genomic era. In the Creator system, an ORF introduced into a donor vector can be transferred with Cre recombinase to a library of acceptor vectors optimized for different applications. Usability of the Creator system is impacted by the ability to easily manipulate DNA, the number of acceptor vectors for downstream applications, and the level of protein expression from Creator vectors. RESULTS: To date, we have developed over 20 novel acceptor vectors that employ a variety of promoters and epitope tags commonly employed for proteomics applications and gene function analysis. We also made several enhancements to the donor vectors including addition of different multiple cloning sites to allow shuttling from pre-existing vectors and introduction of the lacZ alpha reporter gene to allow for selection. Importantly, in order to ameliorate any effects on protein expression of the loxP site between a 5' tag and ORF, we introduced a splicing event into our expression vectors. The message produced from the resulting 'Creator Splice' vector undergoes splicing in mammalian systems to remove the loxP site. Upon analysis of our Creator Splice constructs, we discovered that protein expression levels were also significantly increased. CONCLUSION: The development of new donor and acceptor vectors has increased versatility during the cloning process and made this system compatible with a wider variety of downstream applications. The modifications introduced in our Creator Splice system were designed to remove extraneous sequences due to recombination but also aided in downstream analysis by increasing protein expression levels. As a result, we can now employ epitope tags that are detected less efficiently and reduce our assay scale to allow for higher throughput. The Creator Splice system appears to be an extremely useful tool for proteomics.

Systematic discovery of in vivo phosphorylation networks.

Protein kinases control cellular decision processes by phosphorylating specific substrates. Thousands of in vivo phosphorylation sites have been identified, mostly by proteome-wide mapping. However, systematically matching these sites to specific kinases is presently infeasible, due to limited specificity of consensus motifs, and the influence of contextual factors, such as protein scaffolds, localization, and expression, on cellular substrate specificity. We have developed an approach (NetworKIN) that augments motif-based predictions with the network context of kinases and phosphoproteins. The latter provides 60%-80% of the computational capability to assign in vivo substrate specificity. NetworKIN pinpoints kinases responsible for specific phosphorylations and yields a 2.5-fold improvement in the accuracy with which phosphorylation networks can be constructed. Applying this approach to DNA damage signaling, we show that 53BP1 and Rad50 are phosphorylated by CDK1 and ATM, respectively. We describe a scalable strategy to evaluate predictions, which suggests that BCLAF1 is a GSK-3 substrate.

A Rich1/Amot complex regulates the Cdc42 GTPase and apical-polarity proteins in epithelial cells.

Using functional and proteomic screens of proteins that regulate the Cdc42 GTPase, we have identified a network of protein interactions that center around the Cdc42 RhoGAP Rich1 and organize apical polarity in MDCK epithelial cells. Rich1 binds the scaffolding protein angiomotin (Amot) and is thereby targeted to a protein complex at tight junctions (TJs) containing the PDZ-domain proteins Pals1, Patj, and Par-3. Regulation of Cdc42 by Rich1 is necessary for maintenance of TJs, and Rich1 is therefore an important mediator of this polarity complex. Furthermore, the coiled-coil domain of Amot, with which it binds Rich1, is necessary for localization to apical membranes and is required for Amot to relocalize Pals1 and Par-3 to internal puncta. We propose that Rich1 and Amot maintain TJ integrity by the coordinate regulation of Cdc42 and by linking specific components of the TJ to intracellular protein trafficking.

Strain-dependent role of BrkA during Bordetella pertussis infection of the murine respiratory tract.

Bordetella pertussis, the causative agent of whooping cough, expresses many virulence factors believed to be involved in infection and disease progression. While these factors as a group are required for infection, deletion of individual virulence factor genes generally has limited effects on the ability of B. pertussis to efficiently infect the respiratory tract of mice, suggesting they may perform noncritical or redundant functions. We have recently observed that a B. pertussis strain, putatively with a mutation of a single gene, brkA, results in a severe defect in vivo. Although BrkA has been shown to be required for B. pertussis to resist complement-mediated killing in vitro, the relevance of these findings to the in vivo role of BrkA during infection has not been examined. Transducing this mutation into multiple wild-type B. pertussis strains allowed us to confirm the in vitro phenotype of reduced resistance to serum complement. All DeltabrkA mutants were increased in their sensitivity to complement in vitro, both in the presence and absence of antibodies. However, these strains differed substantially in their phenotypes in vivo. DeltabrkA mutants of recent clinical isolates were indistinguishable from wild-type strains in their efficient infection of respiratory organs, suggesting that the function of BrkA in these strains is noncritical or redundant. In contrast, multiple DeltabrkA strains derived from Tohama I were severely defective during the first week postinoculation compared to their wild-type parent. This defect was present even in complement-deficient mice, revealing a complement-independent phenotype for the DeltabrkA mutant in respiratory tract infection.

Toll-like receptor 4-dependent early elicited tumor necrosis factor alpha expression is critical for innate host defense against Bordetella bronchiseptica.

Toll-like receptor 4 (TLR4) mediates the response to lipopolysaccharide, and its activation induces the expression of a large number of inflammatory genes, many of which are also induced by other pathogen-associated molecular patterns. Interestingly, the subset of genes that are dependent on TLR4 for optimal expression during gram-negative bacterial infection has not been determined. We have previously shown that TLR4-deficient mice rapidly develop acute pneumonia after inoculation with Bordetella bronchiseptica, suggesting that TLR4 is required for expression of early elicited gene products in this model. Microarray analysis with macrophages derived from wild-type and TLR4-deficient mice was used to identify genes whose expression, within 1 h of bacterial exposure, is dependent on TLR4. The results of this investigation suggest that TLR4 is not required for the majority of the transcriptional response to B. bronchiseptica. However, early tumor necrosis factor alpha (TNF-alpha) mRNA expression is primarily dependent on TLR4 and in vitro and in vivo protein levels substantiate this finding. TLR4-deficient mice and TNF-alpha-/- mice are similarly susceptible to infection with relatively low doses of B. bronchiseptica and in vivo neutralization studies indicate that it is the TLR4-dependent early elicited TNF-alpha response that is critical for preventing severe pneumonia and limiting bacterial growth. These results suggest that one critical role for TLR4 is the generation of a robust but transient TNF-alpha response that is critical to innate host defense during acute gram-negative respiratory infection.