Estimation of Sustainable Mortality Thresholds for Grizzly Bears in the Northern Continental Divide Ecosystem

Habitat management and limits on mortality have led to population growth and sizable range expansion for the federally-listed grizzly bear population in the Northern Continental Divide Ecosystem (NCDE), Montana.  Human-caused mortality has coincidentally increased, but it is not clear what level of human-caused mortality would cause the population to decline. A record of annual documented mortalities of independent (?2 years old) bears is maintained for the NCDE, from which an estimate of the total number of mortalities is generated.  Our goal was to estimate sustainable survival rates for independent bears and to develop realistic thresholds for sustainable mortality, which could be applied to these annual estimates.  We estimated survival and recruitment rates using 662 bear-years of telemetry data, performed stochastic modeling, and estimated the annual growth rate as 1.023 and annual population size as 765–960 during 2004–2014.  We then evaluated minimum independent survival rates consistent with a stable to increasing trend, and integrated these sustainable rates with model-estimated population size and mean estimates of total annual independent bear mortality to establish mortality thresholds.  During 2004–2014, estimates of total annual mortality were highly variable, but averaged 13.8 for females and 16.4 for males.  For females and males, respectively, these estimates accounted for only 69% (range 28–168%) and 62% (28–121%) of sustainable mortality thresholds, indicating that approximately 6 and 10 additional annual mortalities could have been sustained without the population declining.  Application and periodic reevaluation of mortality thresholds will help managers reach or maintain a target population size for grizzly bears in the NCDE

Grizzly Bear Population Vital Rates and Trend in the Northern Continental Divide Ecosystem, Montana

We estimated grizzly bear (Ursus arctos) population vital rates and trend for the Northern Continental Divide Ecosystem (NCDE), Montana, between 2004 and 2009 by following radio-collared females and observing their fate and reproductive performance. Our estimates of dependent cub and yearling survival were 0.612 (95% CI = 0.300–0.818) and 0.682 (95% CI = 0.258–0.898). Our estimates of subadult and adult female survival were 0.852 (95% CI = 0.628–0.951) and 0.952 (95% CI = 0.892– 0.980). From visual observations, we estimated a mean litter size of 2.00 cubs/litter. Accounting for cub mortality prior to the first observations of litters in spring, our adjusted mean litter size was 2.27 cubs/litter. We estimated the probabilities of females transitioning from one reproductive state to another between years. Using the stable state probability of 0.322 (95% CI = 0.262–0.382) for females with cub litters, our adjusted fecundity estimate (mx) was 0.367 (95% CI = 0.273–0.461). Using our derived rates, we estimated that the population grew at a mean annual rate of approximately 3% (λ = 1.0306, 95% CI = 0.928–1.102), and 71.5% of 10,000 Monte Carlo simulations produced estimates of λ \u3e 1.0. Our results indicate an increasing population trend of grizzly bears in the NCDE. Coupled with concurrent studies of population size, we estimate that over 1,000 grizzly bears reside in and adjacent to this recovery area. We suggest that monitoring of population trend and other vital rates using radioed females be continued

Grizzly Bear Population Vital Rates and Trend in the Northern Continental Divide Ecosystem, Montana

We estimated grizzly bear (Ursus arctos) population vital rates and trend for the Northern Continental Divide Ecosystem (NCDE), Montana, between 2004 and 2009 by following radio-collared females and observing their fate and reproductive performance. Our estimates of dependent cub and yearling survival were 0.612 (95% CI = 0.300–0.818) and 0.682 (95% CI = 0.258–0.898). Our estimates of subadult and adult female survival were 0.852 (95% CI = 0.628–0.951) and 0.952 (95% CI = 0.892– 0.980). From visual observations, we estimated a mean litter size of 2.00 cubs/litter. Accounting for cub mortality prior to the first observations of litters in spring, our adjusted mean litter size was 2.27 cubs/litter. We estimated the probabilities of females transitioning from one reproductive state to another between years. Using the stable state probability of 0.322 (95% CI = 0.262–0.382) for females with cub litters, our adjusted fecundity estimate (mx) was 0.367 (95% CI = 0.273–0.461). Using our derived rates, we estimated that the population grew at a mean annual rate of approximately 3% (λ = 1.0306, 95% CI = 0.928–1.102), and 71.5% of 10,000 Monte Carlo simulations produced estimates of λ \u3e 1.0. Our results indicate an increasing population trend of grizzly bears in the NCDE. Coupled with concurrent studies of population size, we estimate that over 1,000 grizzly bears reside in and adjacent to this recovery area. We suggest that monitoring of population trend and other vital rates using radioed females be continued

Selecting Cells for Bioartificial Liver Devices and the Importance of a 3D Culture Environment: A Functional Comparison between the HepaRG and C3A Cell Lines

Recently, the first clinical trials on Bioartificial Livers (BALs) loaded with a proliferative human hepatocyte cell source have started. There are two cell lines that are currently in an advanced state of BAL development; HepaRG and HepG2/C3A. In this study we aimed to compare both cell lines on applicability in BALs and to identify possible strategies for further improvement. We tested both cell lines in monolayer- and BAL cultures on growth characteristics, hepatic differentiation, nitrogen-, carbohydrate-, amino acid- and xenobiotic metabolism. Interestingly, both cell lines adapted the hepatocyte phenotype more closely when cultured in BALs; e.g. monolayer cultures produced lactate, while BAL cultures showed diminished lactate production (C3A) or conversion to elimination (HepaRG), and urea cycle activity increased upon BAL culturing in both cell lines. HepaRG-BALs outperformed C3A-BALs on xenobiotic metabolism, ammonia elimination and lactate elimination, while protein synthesis was comparable. In BAL cultures of both cell lines ammonia elimination correlated positively with glutamine production and glutamate consumption, suggesting ammonia elimination was mainly driven by the balance between glutaminase and glutamine synthetase activity. Both cell lines lacked significant urea cycle activity and both required multiple culture weeks before reaching optimal differentiation in BALs. In conclusion, culturing in BALs enhanced hepatic functionality of both cell lines and from these, the HepaRG cells are the most promising proliferative cell source for BAL applicatio

Scaling-up of a HepaRG progenitor cell based bioartificial liver: optimization for clinical application and transport

Anew generation of bioartificial livers, based on differentiated proliferative hepatocyte sources, has been developed. Several practicable and regulatory demands have to be addressed before these can be clinically evaluated. Weidentified three main hurdles: (1) expansion and preservation of the biocomponent, (2) development of scaled-up culture conditions and (3) transport of the device to the bedside. In this study we address these three issues for the HepaRG-progenitor cell line-loaded AMC-Bioartificial Liver. (1) HepaRG cells were expanded in large quantities and then cryopreserved or loaded directly into bioreactors. After 3 weeks of culture, key hepatic functions (ammonia/lactate elimination, apolipoprotein A1 synthesis and cytochrome P450 3A4 activity) did not differ significantly between the two groups. (2) Bioartificial livers were scaled up from 9ml to 540 ml priming volume, with preservation of normalized hepatic functionality. Quantification of amino acid consumption revealed rapid depletion of several amino acids. (3) Whole-device cryopreservation and cooled preservation induced significant loss of hepatic functionality, whereas simulated transport from culture-facility to the bedside in a clinical-grade transport unit with controlled temperature maintenance, medium perfusion and gas supply did not affect functionality. In addition, we assessed tumorigenicity of HepaRG cells in immune-incompetent mice and found no tumor formation of HepaRG cells (n = 12), while HeLa cells induced formation of carcinomas in eight out of 12 mice in 140 day

Oxygen drives hepatocyte differentiation and phenotype stability in liver cell lines

The in vitro generation of terminally differentiated hepatocytes is an unmet need. We investigated the contribution of oxygen concentration to differentiation in human liver cell lines HepaRG and C3A. HepaRG cells were cultured under hypoxia (5%O2), normoxia (21%O2) or hyperoxia (40%O2). Cultures were analysed for hepatic functions, gene transcript levels, and protein expression of albumin, hepatic transcription factor CEBPα, hepatic progenitor marker SOX9, and hypoxia inducible factor (HIF)1α. C3A cells were analysed after exposure to normoxia or hyperoxia. In hyperoxic HepaRG cultures, urea cycle activity, bile acid synthesis, CytochromeP450 3A4 (CYP3A4) activity and ammonia elimination were 165-266% increased. These effects were reproduced in C3A cells. Whole transcriptome analysis of HepaRG cells revealed that 240 (of 23.223) probes were differentially expressed under hyperoxia, with an overrepresentation of genes involved in hepatic differentiation, metabolism and extracellular signalling. Under hypoxia, CYP3A4 activity and ammonia elimination were inhibited almost completely and 5/5 tested hepatic genes and 2/3 tested hepatic transcription factor genes were downregulated. Protein expression of SOX9 and HIF1α was strongly positive in hypoxic cultures, variable in normoxic cultures and predominantly negative in hyperoxic cultures. Conversely, albumin and CEBPα expression were highest in hyperoxic cultures. HepaRG cells that were serially passaged under hypoxia maintained their capacity to differentiate under normoxia, in contrast to cells passaged under normoxia. Hyperoxia increases hepatocyte differentiation in HepaRG and C3A cells. In contrast, hypoxia maintains stem cell characteristics and inhibits hepatic differentiation of HepaRG cells, possibly through the activity of HIF1