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Human primary hepatocytes (PHs) are critical to studying liver functions, drug metabolism and toxicity. PHs isolated from livers that are unacceptable for transplantation have limited expansion and culture viability in vitro, in addition to rapidly deteriorating enzymatic functions. The unsuitability of immortalized hepato-carcinoma cell lines for this function has prompted studies to develop hepatocyte-like cells from alternative sources like ESC, iPS, and other stem cell types using differentiation protocols. This study describes a novel technique to produce expandable and functional hepatocyte-like cells from the fusion of an immortalized human umbilical cord blood derived cell line (E12 MLPC) to normal human primary hepatocytes. Multi-lineage progenitor cells (MLPC) comprise a small subset of mesenchymal-like cells isolated from human umbilical cord blood. MLPC are distinguishable from other mesenchymal-like cells by their extended expansion capacity (up to 80 cell doublings before senescence) and the ability to be differentiated into cells representative of endo-, meso- and ectodermal origins. Transfection of MLPC with the gene for telomerase reverse transcriptase (TERT) resulted in clonal cell lines that were capable of differentiation to different cellular outcomes while maintaining their functional immortality. A methodology for the development of immortalized hepatocyte-like hybrid cells by the in vitro fusion of human MLPC with normal human primary hepatocytes is reported. The resultant hybrid cells exhibited homology with hepatocytes by morphology, immunohistochemistry, urea and albumin production and gene expression. A medium that allows stable long-term expansion of hepatocyte-like fusion cells is described.

Preserving avian flyway connectivity has long been challenged by our capacity to meaningfully quantify continental habitat dynamics and bird movements at temporal and spatial scales underlying long‐distance migrations. Waterbirds migrating hundreds or thousands of kilometers depend on networks of wetland stopover sites to rest and refuel. Entire populations may rely on discrete wetland habitats, particularly in arid landscapes where the loss of limited stopover options can have disproportionately high impacts on migratory cost. Here, we examine flyway connectivity in water‐limited ecosystems of western North America using 108 GPS tagged greater sandhill cranes. Bird movements were used to reconstruct wetland stopover networks across three geographically unique sub‐populations spanning 12 U.S.–Mexican states and Canadian provinces. Networks were monitored with remote sensing to identify long‐term (1988–2019) trends in wetland and agricultural resources supporting migration and evaluated using network theory and centrality metrics as a measure of stopover site importance to flyway connectivity. Sandhill crane space use was analyzed in stopover locations to identify important ownership and landscape factors structuring bird distributions. Migratory efficiency was the primary mechanism underpinning network function. A small number of key stopover sites important to minimizing movement cost between summering and wintering locations were essential to preserving flyway connectivity. Localized efficiencies were apparent in stopover landscapes given prioritization of space use by birds where the proximity of agricultural food resources and flooded wetlands minimized daily movements. Model depictions showing wetland declines from 16% to 18% likely reflect a new normal in landscape drying that could decouple agriculture–waterbird relationships as water scarcity intensifies. Sustaining network resilience will require conservation strategies to balance water allocations preserving agricultural and wetlands on private lands that accounted for 67–96% of habitat use. Study outcomes provide new perspectives of agroecological relationships supporting continental waterbird migration needed to prioritize conservation of landscapes vital to maintaining flyway connectivity.

Quantifying relationships between animal behavior and habitat use is essential to understanding animal decision-making. High-resolution location and acceleration data allows unprecedented insights into animal movement and behavior. These data types allow researchers to study the complex linkages between behavioral plasticity and habitat distribution. We used a novel Markov model in a Bayesian framework to quantify the influence of behavioral state frequencies and environmental variables on transitions among landcover types through joint use of location and tri-axial accelerometer data. Data were collected from 56 greater white-fronted geese ( Anser albifrons frontalis ) across seven ecologically distinct winter regions over two years in midcontinent North America. We showed that goose decision-making varied across landcover types, ecoregions, and abiotic conditions, and was influenced by behavior. We found that time spent in specific behaviors explained variation in the probability of transitioning among habitats, revealing unique behavioral responses from geese among different habitats. Combining GPS and acceleration data allowed unique study of potential influences of an ongoing large-scale range shift in the wintering distribution of a migratory bird across midcontinent North America. We anticipate that behavioral adaptations among variable landscapes is a likely mechanism explaining goose use of highly variable ecosystems during winter in ways which optimize their persistence.

In semi‐arid ecosystems, timing and availability of water is a key uncertainty associated with conservation planning for wetland‐dependent wildlife. Wetlands compose only 1–3% of these landscapes; however, large populations of migratory waterbirds rely on these wetlands to support energetically demanding life history events such as breeding and migration. Migration is considered a crucial period for birds associated with individual survival and reproductive success, yet our understanding of migration ecology remains limited. To better inform conservation planning supportive of these demands, we quantified synchrony of wetland flooding and waterbird migration by reconstructing bi‐monthly surface water patterns from 1984 to 2015 across 11.4 million ha of the semi‐arid Great Basin, USA. Results were then linked to seasonal migration chronologies for seven dabbling ducks species. Seasonal patterns were used in landscape planning simulations to assess efficiency in conservation strategies that aligned temporally sensitive wetland flooding and species migration. Wetland data were combined with land tenure to evaluate periodicity in waterfowl reliance on public and private lands. We found migration chronologies misaligned with wetland flooding. In spring, half (43–59%) to three‐quarters (68–74%) of seasonal wetlands were flooded and available to early‐ and late‐migrating species while seasonal drying restricted wetland flooding to 13–20% of sites during fall migration. Simulations showed wetland conservation inconsiderate of temporal availability was only 67–75% efficient in meeting waterfowl habitat goals on private lands that made up ~70% of flooded wetland area in spring. Private–public wetland flooding was equivalent during fall migration. Accounting for spatiotemporal patterns of wetland flooding is imperative to improving efficiencies linked to migratory bird conservation. Timing of public–private wetland flooding, demonstrated by our models, provides landscape context that emphasized a joint role in supporting migratory waterbird habitat. Integrated management scenarios may capitalize on public lands’ flexibility to expand fall flooding to offset seasonal drying on private lands while targeted incentive‐based conservation assures private wetland flooding in spring. Such scenarios illustrate benefits of holistic public–private wetlands management representing a forward‐looking alternative that aligns conservation with forecasts of increasing water scarcity.

Cysteine-bound hemes are key components of many enzymes and biological sensors. Protonation (deprotonation) of the Cys ligand often accompanies redox transformations of these centers. To characterize these phenomena, we have engineered a series of Thr78Cys/Lys79Gly/Met80X mutants of yeast cytochrome c (cyt c) in which Cys78 becomes one of the axial ligands to the heme. At neutral pH, the protonation state of the coordinated Cys differs for the ferric and ferrous heme species, with Cys binding as a thiolate and a thiol, respectively. Analysis of redox-dependent stability and alkaline transitions of these model proteins, as well as comparisons to Cys binding studies with the minimalist heme peptide microperoxidase-8, demonstrate that the protein scaffold and solvent interactions play important roles in stabilizing a particular Cys–heme coordination. The increased stability of ferric thiolate compared with ferrous thiol arises mainly from entropic factors. This robust cyt c model system provides access to all four forms of Cys-bound heme, including the ferric thiol. Protein motions control the rates of heme redox reactions, and these effects are amplified at low pH, where the proteins are less stable. Thermodynamic signatures and redox reactivity of the model Cys-bound hemes highlight the critical role of the protein scaffold and its dynamics in modulating redox-linked transitions between thiols and thiolates.

During spring, migratory birds are required to optimally balance energetic costs of migration across heterogeneous landscapes and weather conditions to survive and reproduce successfully. Therefore, an individual's migratory performance may influence reproductive outcomes. Given large‐scale changes in land use, climate, and potential carry‐over effects, understanding how individuals migrate in relation to breeding outcomes is critical to predicting how future scenarios may affect populations. We used GPS tracking devices on 56 Greater White‐fronted Geese (Anser albifrons) during four spring migrations to examine whether migration characteristics influenced breeding propensity and breeding outcome. We found a strong longitudinal difference in arrival to the breeding areas (18 days earlier), pre‐nesting duration (90.9% longer), and incubation initiation dates (9 days earlier) between western‐ and eastern‐Arctic breeding regions, with contrasting effects on breeding outcomes, but no migration characteristic strongly influenced breeding outcome. We found that breeding region influenced whether an individual likely pursued a capital or income breeding strategy. Where individuals fell along the capital‐income breeding continuum was influenced by longitude, revealing geographic effects of life‐history strategy among conspecifics. Factors that govern breeding outcomes likely occur primarily upon arrival to breeding areas or are related to individual quality and previous breeding outcome, and may not be directly tied to migratory decision‐making across broad scales. During spring, migratory birds are required to optimally balance energetic costs of migration across heterogeneous landscapes and weather conditions in order to survive and reproduce successfully. Therefore, an individual's migratory performance may influence reproductive outcomes. We used GPS tracking devices on Greater White‐fronted Geese during spring migration to examine whether migration characteristics influenced breeding propensity and breeding outcome. We found highly variable migration strategies led to successful reproductive outcomes, and a geographic gradient of migration characteristics influenced where individuals fell along the capital‐income breeding continuum, revealing geographic effects of life‐history strategy among conspecifics.

Information about species distributions is lacking in many regions of the world, forcing resource managers to answer complex ecological questions with incomplete data. Information gaps are compounded by climate change, driving ecological bottlenecks that can act as new demographic constraints on fauna. Here, we construct greater sandhill crane (Antigone canadensis tabida) summering range in western North America using movement data from 120 GPS‐tagged individuals to determine how landscape composition shaped their distributions. Landscape variables developed from remotely sensed data were combined with bird locations to model distribution probabilities. Additionally, land‐use and ownership were summarized within summer range as a measure of general bird use. Wetland variables identified as important predictors of bird distributions were evaluated in a post hoc analysis to measure long‐term (1984–2022) effects of climate‐driven surface water drying. Wetlands and associated agricultural practices accounted for 1.2% of summer range but were key predictors of occurrence. Bird distributions were structured by riparian floodplains that concentrated wetlands, and flood‐irrigated agriculture in otherwise arid and semi‐arid landscapes. Findings highlighted the role of private lands in greater sandhill crane ecology as they accounted for 78% of predicted distributions. Wetland drying observed in portions of the range from 1984 to 2022 represented an emerging ecological bottleneck that could limit future greater sandhill crane summer range. Study outcomes provide novel insight into the significance of ecosystem services provided by flood‐irrigated agriculture that supported nearly 60% of wetland resources used by birds. Findings suggest greater sandhill cranes function as a surrogate species for agroecology and climate change adaptation strategies seeking to reduce agricultural water use through improved efficiency while also maintaining distinct flood‐irrigation practices supporting greater sandhill cranes and other wetland‐dependent wildlife. We make our wetland and sandhill crane summering distributions available as interactive web‐based mapping tools to inform conservation design. Summer range for greater sandhill cranes (Antigone canadensis tabida) was reconstructed in western North America using movement data from 120 GPS‐tagged individuals to determine how landscape composition shaped their distributions. Wetlands and associated agricultural practices accounted for 1.2% of their range but were key predictors of occurrence. Bird distributions were patterned primarily by riparian floodplains that concentrated wetlands, and flood‐irrigated agriculture in otherwise arid and semi‐arid landscapes. Findings suggest greater sandhill cranes function as an umbrella species for agroecology and climate change adaptation strategies seeking to reduce agricultural water use through improved efficiency while also maintaining distinct flood‐irrigation practices supporting greater sandhill cranes and other wetland‐dependent wildlife.

Managers typically estimate wildlife abundance using surveys within a timeframe that favors increased detectability; however, the ability to account for probabilities of inclusion, detection, and/or presence within a given sampling area is often limited. Cranes provide a good opportunity to research count accuracy because they are large, conspicuous, and often congregate during part of the year, typically on staging areas (i.e., fall and spring) or on wintering grounds. The objectives of this paper are twofold: (1) to evaluate how environmental factors influence crane movement in and out of crane survey areas to identify the best window of availability for annual survey counts; and (2) to evaluate environmental factors that influence overall crane survey counts from year to year. For Objective 1, a generalized linear mixed model was selected to model the probability of crane presence within survey areas using GPS transmitter data. A binary response variable representing crane movement within and outside formal survey areas was used as the dependent variable to investigate environmental covariates that influence movement into survey areas. Probability of crane presence was explained by seven covariates plus a quadratic term for Julian day number. Interactions between Palmer drought severity index (PDSI) and normalized difference vegetation index supported higher probability of crane presence in survey areas during times of drought. Probability of crane presence increased throughout the entire study period (May–October), suggesting that formal surveys in September could be augmented or replaced by surveys in October. For Objective 2, a negative binomial model with linear parameterization was selected to model crane counts using census data compiled from 1995 to 2019. Covariates were acquired at the watershed scale using Hydrologic Unit Code 6 boundaries. Of the 17 covariates investigated, we found that 18‐month precipitation (PPTgss), PDSI, and minimum temperature (Tempmin) explained most of the variability in crane census counts. High PPTgss (antecedent moisture), low PDSI (drought conditions), and low Tempmin (cold extremes) result in higher annual crane counts. The ability to link ecological processes to wildlife movement and population abundance both locally and at landscape scales has long‐ranging implications on resource projections, conservation, and the ability to deploy adaptive management.

River ecosystems in semi-arid environments provide an array of resources that concentrate biodiversity, but also attract human settlement and support economic development. In the southwestern United States, land-use change, drought, and anthropogenic disturbance are compounding factors which have led to departures from historical conditions of river ecosystems, consequently affecting wildlife habitat, including important wintering areas for migratory birds. The Rio Grande (River) in central New Mexico is the lifeblood of the Middle Rio Grande Valley (MRGV), maintaining large urban and agricultural centers and riparian and wetland resources, which disproportionately support a diversity of wildlife. The MRGV has been identified as the most important wintering area for the Rocky Mountain Population of greater sandhill cranes (Antigone canadensis tabida). Presently, however, changes in the hydrogeomorphology of the Rio Grande and landscape modification by humans have reshaped the MRGV and winter habitat for sandhill cranes. To evaluate these impacts, we investigated how land-use practices, anthropogenic disturbance, and river morphology influenced patterns of diurnal and roosting habitat selection by sandhill cranes. During the diurnal period, sandhill cranes relied heavily on managed public lands selecting agriculture crops, such as corn fields, and wetlands for foraging and loafing while avoiding areas with increasing densities of human structures. Sandhill cranes selected areas for roosting in the Rio Grande characterized by shallower water interspersed with sandbars, wide channel width, low bank vegetation, and farther away from disturbances associated with bridges. Our results establish and identify the central processes driving patterns of diel habitat selection by wintering sandhill cranes. Land use and riverine trends have likely gradually reduced winter habitat to managed public lands and limited reaches of the Rio Grande, underscoring the importance of natural resources agencies in supporting migratory birds and challenges involved when managing for wildlife in highly pressured semi-arid environments.

Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) proteins can be designed to bind specified DNA and RNA sequences and hold great promise for the accurate detection of nucleic acids for diagnostics. We integrated commercially available reagents into a CRISPR/Cas9-based lateral flow assay that can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequences with single-base specificity. This approach requires minimal equipment and represents a simplified platform for field-based deployment. We also developed a rapid, multiplex fluorescence CRISPR/Cas9 nuclease cleavage assay capable of detecting and differentiating SARS-CoV-2, influenza A and B, and respiratory syncytial virus in a single reaction. Our findings provide proof-of-principle for CRISPR/Cas9 point-of-care diagnosis as well as a scalable fluorescent platform for identifying respiratory viral pathogens with overlapping symptomology.