Testing foraging optimization models in brown bears: Time for a paradigm shift in nutritional ecology?

How organisms obtain energy to survive and reproduce is fundamental to ecology, yet researchers use theoretical concepts represented by simplified models to estimate diet and predict community interactions. Such simplistic models can sometimes limit our understanding of ecological principles. We used a polyphagous species with a wide distribution, the brown bear (Ursus arctos), to illustrate how disparate theoretical frameworks in ecology can affect conclusions regarding ecological communities. We used stable isotope measurements (δ13 C, δ15 N) from hairs of individually monitored bears in Sweden and Bayesian mixing models to estimate dietary proportions of ants, moose, and three berry species to compare with other brown bear populations. We also developed three hypotheses based on predominant foraging literature, and then compared predicted diets to field estimates. Our three models assumed (1) bears forage to optimize caloric efficiency (optimum foraging model), predicting bears predominately eat berries (~70% of diet) and opportunistically feed on moose (Alces alces) and ants (Formica spp. and Camponotus spp; ~15% each); (2) bears maximize meat intake (maximizing fitness model), predicting a diet of 35%-50% moose, followed by ants (~30%), and berries (~15%); (3) bears forage to optimize macronutrient balance (macronutrient model), predicting a diet of ~22% (dry weight) or 17% metabolizable energy from proteins, with the rest made up of carbohydrates and lipids (~49% and 29% dry matter or 53% and 30% metabolizable energy, respectively). Bears primarily consumed bilberries (Vaccinium myrtillus; 50%-55%), followed by lingonberries (V. vitis-idaea; 22%-30%), crowberries (Empetrum nigrum; 8%-15%), ants (5%-8%), and moose (3%-4%). Dry matter dietary protein was lower than predicted by the maximizing fitness model and the macronutrient balancing model, but protein made up a larger proportion of the metabolizable energy than predicted. While diets most closely resembled predictions from optimal foraging theory, none of the foraging hypotheses fully described the relationship between foraging and ecological niches in brown bears. Acknowledging and broadening models based on foraging theories is more likely to foster novel discoveries and insights into the role of polyphagous species in ecosystems and we encourage this approach.

Bear bile powder alleviates Parkinson's disease-like behavior in mice by inhibiting astrocyte-mediated neuroinflammation.

Parkinson's disease (PD) is a common neurodegenerative disease in middle-aged and elderly people. In particular, increasing evidence has showed that astrocyte-mediated neuroinflammation is involved in the pathogenesis of PD. As a precious traditional Chinese medicine, bear bile powder (BBP) has a long history of use in clinical practice. It has numerous activities, such as clearing heat, calming the liver wind and anti-inflammation, and also exhibits good therapeutic effect on convulsive epilepsy. However, whether BBP can prevent the development of PD has not been elucidated. Hence, this study was designed to explore the effect and mechanism of BBP on suppressing astrocyte-mediated neuroinflammation in a mouse model of PD. PD-like behavior was induced in the mice by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (30 mg·kg-1) for five days, followed by BBP (50, 100, and 200 mg·kg-1) treatment daily for ten days. LPS stimulated rat C6 astrocytic cells were used as a cell model of neuroinflammation. THe results indicated that BBP treatment significantly ameliorated dyskinesia, increased the levels of tyrosine hydroxylase (TH) and inhibited astrocyte hyperactivation in the substantia nigra (SN) of PD mice. Furthermore, BBP decreased the protein levels of glial fibrillary acidic protein (GFAP), cyclooxygenase 2 (COX2) and inducible nitric oxide synthase (iNOS), and up-regulated the protein levels of takeda G protein-coupled receptor 5 (TGR5) in the SN. Moreover, BBP significantly activated TGR5 in a dose-dependent manner, and decreased the protein levels of GFAP, iNOS and COX2, as well as the mRNA levels of GFAP, iNOS, COX2, interleukin (IL) -1ß, IL-6 and tumor necrosis factor-α (TNF-α) in LPS-stimulated C6 cells. Notably, BBP suppressed the phosphorylation of protein kinase B (AKT), inhibitor of NF-κB (IκBα) and nuclear factor-κB (NF-κB) proteins in vivo and in vitro. We also observed that TGR5 inhibitor triamterene attenuated the anti-neuroinflammatory effect of BBP on LPS-stimulated C6 cells. Taken together, BBP alleviates the progression of PD mice by suppressing astrocyte-mediated inflammation via TGR5.

[Efficacy evolution of bear bile and related research on components].

Animal medicine is an important part of traditional Chinese medicine(TCM). Bear bile is one of the rare animal-derived medicinal materials with the functions of clearing the liver, promoting bile secretion, calming the liver, relieving convulsions, clearing heat, and removing toxins. From the Jin Dynasty to the Tang Dynasty, bear bile was mainly used to treat internal diseases, surgical diseases, and pediatric diseases with limitations. At present, bear bile has been used to treat various diseases in pediatrics, gynecology, internal medicine, and surgery. Studies on the chemical constituents and pharmacological effects of bear bile mostly focused on bile acids. Although the non-bile acids also showed certain pharmacological effects, their mechanism of action was less investigated. At present, the source animals of bear bile are national second-class protected animals. Obtaining transformed bear bile powder through biotransformation is expected to alleviate the shortage of bear bile resources to a certain extent. Although related research on bear bile substitutes has protected bear bile resources, there are problems in functional quantification and modern interpretation. It is necessary to sort out the functions and indications of bear bile recorded in ancient books according to related modern research. This study firstly reviewed the evolution of bear bile functions and indications, analyzed the chemical components of bear bile, sorted out the relevant records of the efficacy and clinical application of bear bile in ancient books, and summarized the research progress in the safety of bear bile based on the modern pharmacological effects and clinical applications of bear bile, which is conducive to the clarification of modern efficacy and functional quantification of bear bile and the tentative exploration of the modern interpretation of bear bile.

A novel NADP(H)-dependent 3α-HSDH from the intestinal microbiome of Ursus thibetanus.

3α-HSDHs have a crucial role in the bioconversion of steroids, and have been widely applied in the detection of total bile acid (TBA). In this study, we report a novel NADP(H)-dependent 3α-HSDH (named Sc 3α-HSDH) cloned from the intestinal microbiome of Ursus thibetanus. Sc 3α-HSDH was solubly expressed in E. coli (BL21) as a recombinant glutathione-S-transferase (GST)-tagged protein and freed from its GST-fusion by cleavage using the PreScission protease. Sc 3α-HSDH is a new member of the short-chain dehydrogenases/reductase superfamily (SDRs) with a typical α/ß folding pattern, based on protein three-dimensional models predicted by AlphaFold. The best activity of Sc 3α-HSDH occurred at pH 8.5 and the temperature optima was 55 °C, indicating that Sc 3α-HSDH is not an extremozyme. The catalytic efficiencies (kcat/Km) of Sc 3α-HSDH catalyzing the oxidation reaction with the substrates, glycochenodeoxycholic acid (GCDCA) and glycoursodeoxycholic acid (GUDCA), were 183.617 and 34.458 s-1 mM-1, respectively. In addition, multiple metal ions can enhance the activity of Sc 3α-HSDH when used at concentrations ranging from 2 % to 42 %. The results also suggest that the metagenomic approach is an efficient method for identifying novel enzymes.

Temporal Analysis of Gene Expression and Isoform Switching in Brown Bears (Ursus arctos).

Hibernation in brown bears is an annual process involving multiple physiologically distinct seasons-hibernation, active, and hyperphagia. While recent studies have characterized broad patterns of differential gene regulation and isoform usage between hibernation and active seasons, patterns of gene and isoform expression during hyperphagia remain relatively poorly understood. The hyperphagia stage occurs between active and hibernation seasons and involves the accumulation of large fat reserves in preparation for hibernation. Here, we use time-series analyses of gene expression and isoform usage to interrogate transcriptomic regulation associated with all three seasons. We identify a large number of genes with significant differential isoform usage (DIU) across seasons and show that these patterns of isoform usage are largely tissue-specific. We also show that DIU and differential gene-level expression responses are generally non-overlapping, with only a small subset of multi-isoform genes showing evidence of both gene-level expression changes and changes in isoform usage across seasons. Additionally, we investigate nuanced regulation of candidate genes involved in the insulin signaling pathway and find evidence of hyperphagia-specific gene expression and isoform regulation that may enhance fat accumulation during hyperphagia. Our findings highlight the value of using temporal analyses of both gene- and isoform-level gene expression when interrogating complex physiological phenotypes and provide new insight into the mechanisms underlying seasonal changes in bear physiology.

Isolation and characterization of mesenchymal stem cells from umbilical cord of giant panda.

Umbilical cord-derived mesenchymal stem cells (UC-MSCs) constitute a class of cells with significant self-renewal and multilineage differentiation properties and have great potential for therapeutic applications and the genetic conservation of endangered animals. In this study, we successfully isolated and cultured UC-MSCs from the blood vessels of giant panda umbilical cord (UC). The cells were arranged in a vortex or cluster pattern and exhibited a normal karyotype, showing the morphological characteristics of fibroblasts. In addition, we found that basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) promoted cell proliferation, whereas stem cell factor (SCF) did not promote cell proliferation. Cultured UC-MSCs were negative for CD34 (hematopoietic stem cell marker) and CD31 (endothelial cell marker), but positive for MSC markers (CD44, CD49f, CD105, and CD73) and stem cell markers (KLF4, SOX2, and THY1). Similar to other MSCs, giant panda UC-MSCs have multiple differentiation ability and can differentiate into adipocytes, osteoblasts and chondrocytes. Giant panda UC-MSCs are new resources for basic research as cell models following their differentiation into different cell types and for future clinical treatments of giant panda diseases.

Losing seasonal patterns in a hibernating omnivore? Diet quality proxies and faecal cortisol metabolites in brown bears in areas with and without artificial feeding.

Bears are omnivores particularly well-adapted to variations in the nutritional composition, quality and availability of food resources. Artificial feeding practices have been shown to strongly influence diet composition and seasonality, as well as to cause alterations in wintering and movement in brown bears (Ursus arctos). In this study, we investigated seasonal differences (hypophagia vs hyperphagia) in food quality of two brown bear subpopulations in the Polish Carpathians using faecal nitrogen (FN) and carbon (FC) estimates. The subpopulations inhabit areas that differ in artificial feeding practices: no artificial feeding occurs in the western subpopulation (Tatra Mountains), while artificial food targeted to ungulates is provided and used year-round in the eastern subpopulation (Bieszczady Mountains). We also compared these results with faecal cortisol metabolites (FCM) to explore how FN and FC correlate with the hypothalamic-pituitary-adrenal axis activity and if the seasonal patterns are apparent. We found that in Tatra Mts bears fed on significantly higher quality diet, as shown by FN and FC values, and had significantly higher FC levels in hyperphagia, when they accumulate fat reserves for wintering. The pattern in FCM levels for Tatra subpopulation followed the changes in energy intake during the seasons of hypo- and hyperphagia, while in Bieszczady Mts, the area with intensive feeding, no seasonal patterns could be observed. Artificial feeding practices may disrupt nutrient phenology and seasonality, relative to subpopulations with natural diets. We showed that the availability of human-provided foods may alter not only the overall dietary quality, but also hormonal patterns linked to seasonal nutritional requirements. Combining FN, FC and FCM proved to be a useful tool for reconstructing diet quality and related physiological patterns.

Hibernating bear serum hinders osteoclastogenesis in-vitro.

Bears do not suffer from osteoporosis during hibernation, which is associated with long-term inactivity, lack of food intake, and cold exposure. However, the mechanisms involved in bone loss prevention have scarcely been elucidated in bears. We investigated the effect of serum from hibernating Japanese black bears (Ursus thibetanus japonicus) on differentiation of peripheral blood mononuclear cells (PBMCs) to osteoclasts (OCs). PBMCs collected from 3 bears were separately cultured with 10% serum of 4 active and 4 hibernating bears (each individual serum type was assessed separately by a bear PBMCs), and differentiation were induced by treatment with macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). PBMCs that were cultured with the active bear serum containing medium (ABSM) differentiated to multi-nucleated OCs, and were positive for TRAP stain. However, cells supplemented with hibernating bear serum containing medium (HBSM) failed to form OCs, and showed significantly lower TRAP stain (p < 0.001). On the other hand, HBSM induced proliferation of adipose derived mesenchymal stem cells (ADSCs) similarly to ABSM (p > 0.05), indicating no difference on cell growth. It was revealed that osteoclastogenesis of PBMCs is hindered by HBSM, implying an underlying mechanism for the suppressed bone resorption during hibernation in bears. In addition, this study for the first time showed the formation of bears' OCs in-vitro.

Assessing Urinary Metabolomics in Giant Pandas Using Chromatography/Mass Spectrometry: Pregnancy-Related Changes in the Metabolome.

Giant pandas represent one of the most endangered species worldwide, and their reproductive capacity is extremely low. They have a relatively long gestational period, mainly because embryo implantation is delayed. Giant panda cubs comprise only a small proportion of the mother's body weight, making it difficult to determine whether a giant panda is pregnant. Timely determination of pregnancy contributes to the efficient breeding and management of giant pandas. Meanwhile, metabolomics studies the metabolic composition of biological samples, which can reflect metabolic functions in cells, tissues, and organisms. This work explored the urinary metabolites of giant pandas during pregnancy. A sample of 8 female pandas was selected. Differences in metabolite levels in giant panda urine samples were analyzed via ultra-high-performance liquid chromatography/mass spectrometry comparing pregnancy to anoestrus. Pattern recognition techniques, including partial least squares-discriminant analysis and orthogonal partial least squares-discriminant analysis, were used to analyze multiple parameters of the data. Compared with the results during anoestrus, multivariate statistical analysis of results obtained from the same pandas being pregnant identified 16 differential metabolites in the positive-ion mode and 43 differential metabolites in the negative-ion mode. The levels of tryptophan, choline, kynurenic acid, uric acid, indole-3-acetaldehyde, taurine, and betaine were higher in samples during pregnancy, whereas those of xanthurenic acid and S-adenosylhomocysteine were lower. Amino acid metabolism, lipid metabolism, and organic acid production differed significantly between anoestrus and pregnancy. Our results provide new insights into metabolic changes in the urine of giant pandas during pregnancy, and the differential levels of metabolites in urine provide a basis for determining pregnancy in giant pandas. Understanding these metabolic changes could be helpful for managing pregnant pandas to provide proper nutrients to their fetuses.

Metabolic derangements and reduced survival of bile-extracted Asiatic black bears (Ursus thibetanus).

BACKGROUND: Across China and Southeast Asia, an estimated 17,000 bears are currently farmed for bile, primarily for traditional medicines. Depending on country, bile is extracted daily via transabdominal gallbladder fistulas, indwelling catheters, or needle aspiration. Despite claims that bears do not develop adverse effects from bile extraction, health issues identified in bears removed from bile farms include bile-extraction site infections, abdominal hernias, peritonitis, cholecystitis, hepatic neoplasia, cardiac disease, skeletal abnormalities, and abnormal behaviors. We present a comprehensive assessment of the effects of bile farming by comparing serum biochemical and hematological values of bears from farms that were bile-extracted (BE) and bears from farms not bile-extracted (FNE) with bears from non-farm captive (ZOO) and free-range (FR) environments. We hypothesized BE bears would have significant laboratory abnormalities compared to all non-extracted bear groups. We also hypothesized BE bears would have reduced long-term survival compared to FNE bears despite removal from farms. RESULTS: BE bears exhibited the highest values and greatest variation (on a population level) in laboratory parameters compared to all non-extracted bear groups particularly for alanine transaminase, gamma glutamyltransferase (GGT), total bilirubin (TBIL), alkaline phosphatase (ALKP), blood urea nitrogen, creatinine (CREA), and total white blood cell count. Significant differences were detected between bear groups when accounting for season, sex, and/or age. BE bears exhibited greater mean serum GGT compared to all non-extracted bear groups, and the odds of having elevated TBIL were 7.3 times greater for BE bears, consistent with hepatobiliary disease. Biochemical parameter elevations in BE bears persisted up to 14 years post-rescue, consistent with long-term effects of bile-extraction. BE bears that arrived with elevated CREA and ALKP had median survival times of 1 and 4 years respectively, and regardless of laboratory abnormalities, BE bears had significantly shorter survival times compared to FNE bears. CONCLUSIONS: Our results provide strong evidence that bile extraction practices not only represent a temporary constraint for bears' welfare, but confer distinct long-term adverse health consequences. Routine laboratory panels may be insensitive to detect the extent of underlying illness in BE bears as these bears have significantly reduced survival regardless of biochemical assessment compared to FNE bears.

Pharmacology of the giant panda (Ailuropoda melanoleuca) melanocortin-3 receptor.

The melanocortin-3 receptor (MC3R) is a member of the G protein-coupled receptor superfamily that plays a critical role in controlling energy balance and metabolism. Although pharmacological characterization of MC3R has been reported previously in several other species, there is no report on the MC3R from giant panda (Ailuropoda melanoleuca). This ancient species is known as a 'living fossil' and is among the most endangered animals in the world. Giant panda survive on a specialized diet of bamboo despite possessing a typical carnivorous digestive system. We report herein the molecular cloning and pharmacological characterization of amMC3R. Homology and phylogenetic analysis showed that amMC3R was highly homologous (>85%) to several other mammalian MC3Rs. Using human MC3R (hMC3R) as a control, the binding of five agonists, [Nle4, D-Phe7]-α-melanocyte stimulating hormone (NDP-MSH), α-, ß-, γ-, and D-Trp8-γ-MSH, was investigated, as well as Gs-cAMP and pERK1/2 signaling. The results showed that amMC3R bound NDP- and D-Trp8-γ-MSH with the highest affinity, followed by α-, ß-, and γ-MSH, with the same rank order as hMC3R. When stimulated with agonists, amMC3R displayed increased intracellular cAMP and activation of pERK1/2. These data suggest that the cloned amMC3R was a functional receptor. The availability of amMC3R and knowledge of its pharmacological functions will assist further investigation of its role in controlling energy balance and metabolism.

Soleus muscle stability in wild hibernating black bears.

Based on studies of fast skeletal muscles, hibernating black and brown bears resist skeletal muscle atrophy during months of reduced physical activity and not feeding. The present study examined atrophy sparing in the slow soleus muscle, known to be highly prone to disuse atrophy in humans and other mammals. We demonstrated histochemically that the black bear soleus is rich in slow fibers, averaging 84.0 ± 6.6%. The percentages of slow fibers in fall (87.3 ± 4.9%) and during hibernation (87.1 ± 5.6%) did not differ ( P = 0.3152) from summer. The average fiber cross-sectional area to body mass ratio (48.6 ± 11.7 µm2/kg) in winter hibernating bears was not significantly different from that of summer (54.1 ± 11.8 µm2/kg, P = 0.4186) and fall (47.0 ± 9.7 µm2/kg, P = 0.9410) animals. The percentage of single hybrid fibers containing both slow and fast myosin heavy chains, detected biochemically, increased from 2.6 ± 3.8% in summer to 24.4 ± 24.4% ( P = 0.0244) during hibernation. The shortening velocities of individual hybrid fibers remained unchanged from that of pure slow and fast fibers, indicating low content of the minority myosins. Slow and fast fibers in winter bears exhibited elevated specific tension (kN/m2; 22%, P = 0.0161 and 11%, P = 0.0404, respectively) and maintained normalized power. The relative stability of fiber type percentage and size, fiber size-to-body mass ratio, myosin heavy chain isoform content, shortening velocity, power output, and elevated specific tension during hibernation validates the ability of the black bear to preserve the biochemical and performance characteristics of the soleus muscle during prolonged hibernation.

Combined thin layer chromatography and gas chromatography with mass spectrometric analysis of lipid classes and fatty acids in malnourished polar bears (Ursus maritimus) which swam to Iceland.

Between 2008 and 2011, four polar bears (Ursus maritimus) from the Greenland population swam and/or drifted on ice to Iceland where they arrived in very poor body condition. Body fat resources in these animals were only between 0% and 10% of the body weight (usually 25%). Here we studied the lipid composition in different tissues (adipose tissue if available, liver, kidney and muscle). Lipid classes were determined by thin layer chromatography (TLC) and on-column gas chromatography with mass spectrometry (GC/MS). The fatty acid pattern of total lipids and free fatty acids was analyzed by GC/MS in selected ion monitoring (SIM) mode. Additionally, cholesteryl esters and native fatty acid methyl esters, initially detected as zones in thin layer chromatograms, were enriched by solid phase extraction and quantified by GC/MS. The ratio of free fatty acids to native fatty acid methyl esters could be correlated with the remained body lipids in the polar bears and thus may also serve as a marker for other starving animals or even for humans.

Per- and polyfluoroalkyl substances (PFASs) - New endocrine disruptors in polar bears (Ursus maritimus)?

Per- and polyfluoroalkyl substances (PFASs) are emerging in the Arctic and accumulate in brain tissues of East Greenland (EG) polar bears. In vitro studies have shown that PFASs might possess endocrine disrupting abilities and therefore the present study was conducted to investigate potential PFAS induced alterations in brain steroid concentrations. The concentrations of eleven steroid hormones were determined in eight brain regions from ten EG polar bears. Pregnenolone (PRE), the dominant progestagen, was found in mean concentrations of 5-47ng/g (ww) depending on brain region. PRE showed significantly (p<0.01) higher concentrations in female compared to male bears. Dehydroepiandrosterone (DHEA) found in mean concentrations 0.67-4.58ng/g (ww) was the androgen found in highest concentrations. Among the estrogens estrone (E1) showed mean concentrations of 0.90-2.21ng/g (ww) and was the most abundant. Remaining steroid hormones were generally present in concentrations below 2ng/g (ww). Steroid levels in brain tissue could not be explained by steroid levels in plasma. There was however a trend towards increasing estrogen levels in plasma resulting in increasing levels of androgens in brain tissue. Correlative analyses showed positive associations between PFASs and 17α-hydroxypregnenolone (OH-PRE) (e.g. perflouroalkyl sulfonates (∑PFSA): p<0.01, r=0.39; perfluoroalkyl carboxylates (∑PFCA): p<0.01, r=0.61) and PFCA and testosterone (TS) (∑PFCA: p=0.03, r=0.30) across brain regions. Further when investigating correlative associations in specific brain regions significant positive correlations were found between ∑PFCA and several steroid hormones in the occipital lobe. Correlative positive associations between PFCAs and steroids were especially observed for PRE, progesterone (PRO), OH-PRE, DHEA, androstenedione (AN) and testosterone (TS) (all p≤0.01, r≥0.7). The results from the present study generally indicate that an increase in PFASs concentration seems to concur with an increase in steroid hormones of EG polar bears. It is, however, not possible to determine whether alterations in brain steroid concentrations arise from interference with de novo steroid synthesis or via disruption of peripheral steroidogenic tissues mainly in gonads and feedback mechanisms. Steroids are important for brain plasticity and gender specific behavior as well as postnatal development and sexually dimorph brain function. The present work indicates an urgent need for a better mechanistic understanding of how PFASs may affect the endocrine system of polar bears and potentially other mammal species.

Long-Term Survey of Cadmium and Lead Contamination in Japanese Black Bears Captured in Iwate Prefecture, Japan.

Cadmium and lead were measured in liver and kidney samples of 242 Japanese black bears (Ursus thibetanus japonicus) captured from 1999 to 2014 from two local populations in Japan. The median concentration of cadmium was 0.54 (mean: 0.80) mg/kg-w.w. in liver and 7.7 (mean: 11.8) mg/kg-w.w. in kidney. The median concentration of lead was 0.24 (mean: 0.40) and 0.21 (mean: 0.32) mg/kg-w.w. in liver and kidney, respectively. Bears in the Kita-ou local population had higher concentrations of cadmium and lead than those in the Kitakami Highlands local population. No chronological change was observed in cadmium levels in tissues, but the percentage of bears whose lead levels exceeded 0.5 mg/kg-w.w. has been decreasing in recent years. Countermeasures against lead poisoning in wildlife, which were instituted in 2002, may have contributed to the decrease in lead contamination of the Japanese black bear.

Current-use pesticides in seawater and their bioaccumulation in polar bear-ringed seal food chains of the Canadian Arctic.

The distribution of current-use pesticides (CUPs) in seawater and their trophodynamics were investigated in 3 Canadian Arctic marine food chains. The greatest ranges of dissolved-phase concentrations in seawater for each CUP were endosulfan sulfate (less than method detection limit (MDL) to 19 pg L(-1) ) > dacthal (0.76-15 pg L(-1) ) > chlorpyrifos (less than MDL to 8.1 pg L(-1) ) > pentachloronitrobenzene (less than MDL to 2.6 pg L(-1) ) > α-endosulfan (0.20-2.3 pg L(-1) ). Bioaccumulation factors (BAFs, water-respiring organisms) were greatest in plankton, including chlorothalonil (log BAF = 7.4 ± 7.1 L kg(-1) , mean ± standard error), chlorpyrifos (log BAF = 6.9 ± 6.7 L kg(-1) ), and α-endosulfan (log BAF = 6.5 ± 6.0 L kg(-1) ). The largest biomagnification factors (BMFs) were found for dacthal in the capelin:plankton trophic relationship (BMF = 13 ± 5.0) at Cumberland Sound (Nunvavut), and for ß-endosulfan (BMF = 16 ± 4.9) and α-endosulfan (BMF = 9.3 ± 2.8) in the polar bear-ringed seal relationship at Barrow and Rae Strait (NU), respectively. Concentrations of endosulfan sulfate exhibited trophic magnification (increasing concentrations with increasing trophic level) in the poikilothermic portion of the food web (trophic magnification factor = 1.4), but all of the CUPs underwent trophic dilution in the marine mammal food web, despite some trophic level-specific biomagnification. Together, these observations are most likely indicative of metabolism of these CUPs in mammals. Environ Toxicol Chem 2016;35:1695-1707. © 2016 SETAC.

Seasonal changes in the expression of energy metabolism-related genes in white adipose tissue and skeletal muscle in female Japanese black bears.

Bears undergo annual cycles in body mass: rapid fattening in autumn (i.e., hyperphagia), and mass loss in winter (i.e., hibernation). To investigate how Japanese black bears (Ursus thibetanus japonicus) adapt to such extreme physiological conditions, we analyzed changes in the mRNA expression of energy metabolism-related genes in white adipose tissues and skeletal muscle throughout three physiological stages: normal activity (June), hyperphagia (November), and hibernation (March). During hyperphagia, quantitative real-time polymerase chain reaction analysis revealed the upregulation of de novo lipogenesis-related genes (e.g., fatty acid synthase and diacylglycerol O-acyltransferase 2) in white adipose tissue, although the bears had been maintained with a constant amount of food. In contrast, during the hibernation period, we observed a downregulation of genes involved in glycolysis (e.g., glucose transporter 4) and lipogenesis (e.g., acetyl-CoA carboxylase 1) and an upregulation of genes in fatty acid catabolism (e.g., carnitine palmitoyltransferase 1A) in both tissue types. In white adipose tissues, we observed upregulation of genes involved in glyceroneogenesis, including pyruvate carboxylase and phosphoenolpyruvate carboxykinase 1, suggesting that white adipose tissue plays a role in the recycling of circulating free fatty acids via re-esterification. In addition, the downregulation of genes involved in amino acid catabolism (e.g., alanine aminotransferase) and the TCA cycle (e.g., pyruvate carboxylase) indicated a role of skeletal muscle in muscle protein sparing and pyruvate recycling via the Cori cycle. These examples of coordinated transcriptional regulation would contribute to rapid mass gain during the pre-hibernation period and to energy preservation and efficient energy production during the hibernation period.

Selenium in brown bears (Ursus arctos) from Croatia: Relation to cadmium and mercury.

Muscle (n = 111), liver (n = 111), and kidney cortex (n = 101) samples from brown bears (Ursus arctos) were collected in the 2009 and 2010 hunting seasons in Croatia and analysed for selenium (Se), cadmium (Cd), and total mercury (Hg). The aim was to assess the levels of these elements according to age, sex, and season of collection, and to investigate possible Se/Cd and Se/Hg interactions. Median Se concentrations were 0.139 µg/g in muscle, 0.409 µg/g in liver and 1.75 µg/g wet mass in kidney cortex. Median Cd and Hg were 0.0078 and 0.0018 µg/g in muscle, 1.09 and 0.031 µg/g in liver, and 16.5 and 0.206 µg/g wet mass in the renal cortex, respectively. Se/Cd molar ratios were less than 1 in the kidney cortex, and close to or above 1 in liver and muscle, respectively. Toxic Cd and Hg correlated with Se in all of the studied tissues. Sex differences were found for all three elements (except Se in liver), with females having higher tissue concentration than males. Only Cd showed age-dependence. Bear samples collected in fall had higher Se in muscles, and Hg in muscles and liver compared to samples collected in spring. Element concentrations in brown bear tissues were within the range of previously reported studies. Bear meat is considered a rich source of Se, safe for consumption with regard to its Cd and Hg content. According to the molar ratio and correlation results, we assume that Se binding is not the primary detoxification pathway for Cd and Hg in brown bears.

Hydrogen sulfide and nitric oxide metabolites in the blood of free-ranging brown bears and their potential roles in hibernation.

During winter hibernation, brown bears (Ursus arctos) lie in dens for half a year without eating while their basal metabolism is largely suppressed. To understand the underlying mechanisms of metabolic depression in hibernation, we measured type and content of blood metabolites of two ubiquitous inhibitors of mitochondrial respiration, hydrogen sulfide (H2S) and nitric oxide (NO), in winter-hibernating and summer-active free-ranging Scandinavian brown bears. We found that levels of sulfide metabolites were overall similar in summer-active and hibernating bears but their composition in the plasma differed significantly, with a decrease in bound sulfane sulfur in hibernation. High levels of unbound free sulfide correlated with high levels of cysteine (Cys) and with low levels of bound sulfane sulfur, indicating that during hibernation H2S, in addition to being formed enzymatically from the substrate Cys, may also be regenerated from its oxidation products, including thiosulfate and polysulfides. In the absence of any dietary intake, this shift in the mode of H2S synthesis would help preserve free Cys for synthesis of glutathione (GSH), a major antioxidant found at high levels in the red blood cells of hibernating bears. In contrast, circulating nitrite and erythrocytic S-nitrosation of glyceraldehyde-3-phosphate dehydrogenase, taken as markers of NO metabolism, did not change appreciably. Our findings reveal that remodeling of H2S metabolism and enhanced intracellular GSH levels are hallmarks of the aerobic metabolic suppression of hibernating bears.

Overexpression, purification, molecular characterization and the effect on tumor growth of ribosomal protein L22 from the Giant Panda (Ailuropoda melanoleuca).

The ribosomal protein L22 (RPL22) protein belongs to the L22E family of ribosomal proteins. It is located in the cytoplasm. The purpose of this paper was to explore the structure and anti-cancer function of RPL22 of the Giant Panda (Ailuropoda melanoleuca). The cDNA of RPL22 was cloned successfully from the Giant Panda using RT-PCR technology. We constructed a recombinant expression vector containing RPL22 cDNA and over-expressed it in Escherichia coli using pET28a plasmids. The expression product obtained was purified by using Ni chelating affinity chromatography. The result indicated that the length of the fragment cloned is 414 bp, and it contains an open-reading frame of 387 bp encoding 128 amino acids. Primary structure analysis revealed that the molecular weight of the putative RPL22 protein is 14.74 kDa with a theoretical pI 9.21. The RPL22 gene can be really expressed in E. coli and the RPL22 protein, fusioned with the N-terminally His-tagged protein, gave rise to the accumulation of an expected 20.1 kDa polypeptide. The data showed that the recombinant protein RPL22 had a time- and dose-dependency on the cell growth inhibition rate. The human laryngeal carcinoma Hep-2 cells treated with 0.05-6 µg/ml of RPL22 for 24 h displayed significant cell growth inhibition (p<0.05, n=8) in assayed using MTT compared to the control (untreated) cells. The data indicate that the effect at low concentrations is better than high concentrations, and the concentration of 1.5 µg/ml has the best rate of growth inhibition of 47.70%. The inhibitory rate in mice treated with 1.5 µg/ml RPL22 protein can reach 43.75%. Histology of tumor organs shows that the tissues arranged looser in RPL22 group than those in control group. Meanwhile, there is no obvious damage to other organs, such as heart, lung and kidney. Further research is on going to determine the bioactive principle(s) of recombinant protein RPL22 responsible for its anticancer activity.