Root nodules of red alder (Alnus rubra) and sitka alder (Alnus viridis ssp. sinuata) are inhabited by taxonomically diverse cultivable microbial endophytes.

The root nodules of actinorhizal plants are home to nitrogen-fixing bacterial symbionts, known as Frankia, along with a small percentage of other microorganisms. These include fungal endophytes and non-Frankia bacteria. The taxonomic and functional diversity of the microbial consortia within these root nodules is not well understood. In this study, we surveyed and analyzed the cultivable, non-Frankia fungal and bacterial endophytes of root nodules from red and Sitka alder trees that grow together. We examined their taxonomic diversity, co-occurrence, differences between hosts, and potential functional roles. For the first time, we are reporting numerous fungal endophytes of alder root nodules. These include Sporothrix guttuliformis, Fontanospora sp., Cadophora melinii, an unclassified Cadophora, Ilyonectria destructans, an unclassified Gibberella, Nectria ramulariae, an unclassified Trichoderma, Mycosphaerella tassiana, an unclassified Talaromyces, Coniochaeta sp., and Sistotrema brinkmanii. We are also reporting several bacterial genera for the first time: Collimonas, Psychrobacillus, and Phyllobacterium. Additionally, we are reporting the genus Serratia for the second time, with the first report having been recently published in 2023. Pseudomonas was the most frequently isolated bacterial genus and was found to co-inhabit individual nodules with both fungi and bacteria. We found that the communities of fungal endophytes differed by host species, while the communities of bacterial endophytes did not.

FicD regulates adaptation to the unfolded protein response in the murine liver.

The unfolded protein response (UPR) is a cellular stress response that is activated when misfolded proteins accumulate in the endoplasmic reticulum (ER). Regulation of the UPR response must be adapted to the needs of the cell as prolonged UPR responses can result in disrupted cellular function and tissue damage. Previously, we discovered that the enzyme FicD (also known as Fic or HYPE) through its AMPylation and deAMPylation activity can modulate the UPR response via post-translational modification of BiP. FicD AMPylates BiP during homeostasis and deAMPylates BiP during stress. We hypothesized that FicD regulation of the UPR will play a role in mitigating the deleterious effects of UPR activation in tissues with frequent physiological stress. Here, we explore the role of FicD in the murine liver. As seen in our pancreatic studies, livers lacking FicD exhibit enhanced UPR signaling in response to short term physiologic fasting and feeding stress. However, in contrast to studies on the pancreas, livers, as a more regenerative tissue, remained remarkably resilient in the absence of FicD. The livers of FicD-/- did not show marked changes in UPR signaling or damage after either chronic high fat diet (HFD) feeding or acute pathological UPR induction. Intriguingly, FicD-/- mice showed changes in UPR induction and weight loss patterns following repeated pathological UPR induction. These findings indicate that FicD regulates UPR responses during mild physiological stress and in adaptation to repeated stresses, but there are tissue specific differences in the requirement for FicD regulation.

FicD regulates adaptation to the unfolded protein response in the murine liver.

The unfolded protein response (UPR) is a cellular stress response that is activated when misfolded proteins accumulate in the endoplasmic reticulum (ER). The UPR elicits a signaling cascade that results in an upregulation of protein folding machinery and cell survival signals. However, prolonged UPR responses can result in elevated cellular inflammation, damage, and even cell death. Thus, regulation of the UPR response must be tuned to the needs of the cell, sensitive enough to respond to the stress but pliable enough to be stopped after the crisis has passed. Previously, we discovered that the bi-functional enzyme FicD can modulate the UPR response via post-translational modification of BiP. FicD AMPylates BiP during homeostasis and deAMPylates BiP during stress. We found this activity is important for the physiological regulation of the exocrine pancreas. Here, we explore the role of FicD in the murine liver. Like our previous studies, livers lacking FicD exhibit enhanced UPR signaling in response to short term physiologic fasting and feeding stress. However, the livers of FicD -/- did not show marked changes in UPR signaling or damage after either chronic high fat diet (HFD) feeding or acute pathological UPR induction. Intriguingly, FicD -/- mice showed changes in UPR induction and weight loss patterns following repeated pathological UPR induction. These findings show that FicD regulates UPR responses during mild physiological stress and may play a role in maintaining resiliency of tissue through adaptation to repeated ER stress.

FicD Sensitizes Cellular Response to Glucose Fluctuations in Mouse Embryonic Fibroblasts.

During homeostasis, the endoplasmic reticulum (ER) maintains productive transmembrane and secretory protein folding that is vital for proper cellular function. The ER-resident HSP70 chaperone, BiP, plays a pivotal role in sensing ER stress to activate the unfolded protein response (UPR). BiP function is regulated by the bifunctional enzyme FicD that mediates AMPylation and deAMPylation of BiP in response to changes in ER stress. AMPylated BiP acts as a molecular rheostat to regulate UPR signaling, yet little is known about the molecular consequences of FicD loss. In this study, we investigate the role of FicD in mouse embryonic fibroblast (MEF) response to pharmacologically and metabolically induced ER stress. We find differential BiP AMPylation signatures when comparing robust chemical ER stress inducers to physiological glucose starvation stress and recovery. Wildtype MEFs respond to pharmacological ER stress by downregulating BiP AMPylation. Conversely, BiP AMPylation in wildtype MEFs increases upon metabolic stress induced by glucose starvation. Deletion of FicD results in widespread gene expression changes under baseline growth conditions. In addition, FicD null MEFs exhibit dampened UPR signaling, altered cell stress recovery response, and unconstrained protein secretion. Taken together, our findings indicate that FicD is important for tampering UPR signaling, stress recovery, and the maintenance of secretory protein homeostasis. Significance Statement: The chaperone BiP plays a key quality control role in the endoplasmic reticulum, the cellular location for the production, folding, and transport of secreted proteins. The enzyme FicD regulates BiP's activity through AMPylation and deAMPylation. Our study unveils the importance of FicD in regulating BiP and the unfolded protein response (UPR) during stress. We identify distinct BiP AMPylation signatures for different stressors, highlighting FicD's nuanced control. Deletion of FicD causes widespread gene expression changes, disrupts UPR signaling, alters stress recovery, and perturbs protein secretion in cells. These observations underscore the pivotal contribution of FicD for preserving secretory protein homeostasis. Our findings deepen the understanding of FicD's role in maintaining cellular resilience and open avenues for therapeutic strategies targeting UPR-associated diseases.

Extracellular vesicles and insulin-mediated vascular function in metabolic syndrome.

Metabolic Syndrome (MetS) raises cardiovascular disease risk. Extracellular vesicles (EVs) have emerged as important mediators of insulin sensitivity, although few studies on vascular function exist in humans. We determined the effect of insulin on EVs in relation to vascular function. Adults with MetS (n = 51, n = 9 M, 54.8 ± 1.0 years, 36.4 ± 0.7 kg/m2 , ATPIII: 3.5 ± 0.1 a.u., VO2 max: 22.1 ± 0.6 ml/kg/min) were enrolled in this cross-sectional study. Peripheral insulin sensitivity (M-value) was determined during a euglycemic clamp (40 mU/m2 /min, 90 mg/dl), and blood was collected for EVs (CD105+, CD45+, CD41+, TX+, and CD31+; spectral flow cytometry), inflammation, insulin, and substrates. Central hemodynamics (applanation tonometry) was determined at 0 and 120 min via aortic waveforms. Pressure myography was used to assess insulin-induced arterial vasodilation from mouse 3rd order mesenteric arteries (100-200 µm in diameter) at 0.2, 2 and 20 nM of insulin with EVs from healthy and MetS adults. Adults with MetS had low peripheral insulin sensitivity (2.6 ± 0.2 mg/kg/min) and high HOMA-IR (4.7 ± 0.4 a.u.) plus Adipose-IR (13.0 ± 1.3 a.u.). Insulin decreased total/particle counts (p < 0.001), CD45+ EVs (p = 0.002), AIx75 (p = 0.005) and Pb (p = 0.04), FFA (p < 0.001), total adiponectin (p = 0.006), ICAM (p = 0.002), and VCAM (p = 0.03). Higher M-value related to lower fasted total EVs (r = -0.40, p = 0.004) while higher Adipose-IR associated with higher fasted EVs (r = 0.42, p = 0.004) independent of VAT. Fasting CD105+ and CD45+ derived total EVs correlated with fasting AIx75 (r = 0.29, p < 0.05) and Pb (r = 0.30, p < 0.05). EVs from MetS participants blunted insulin-induced vasodilation in mesenteric arteries compared with increases from healthy controls across insulin doses (all p < 0.005). These data highlight EVs as potentially novel mediators of vascular insulin sensitivity and disease risk.

Stable coexistence or competitive exclusion? Fern endophytes demonstrate rapid turnover favouring a dominant fungus.

Fungal endophytes are critical members of the plant microbiome, but their community dynamics throughout an entire growing season are underexplored. Additionally, most fungal endophyte research has centred on seed-reproducing hosts, while spore-reproducing plants also host endophytes and may be colonized by unique community members. In order to examine annual fungal endophyte community dynamics in a spore-reproducing host, we explored endophytes in a single population of ferns, Polystichum munitum, in the Pacific Northwest. Through metabarcoding, we characterized the community assembly and temporal turnover of foliar endophytes throughout a growing season. From these results, we selected endophytes with outsized representations in sequence data and performed in vitro competition assays. Finally, we inoculated sterile fern gametophytes with dominant fungi observed in the field and determined their effects on host performance. Sequencing demonstrated that ferns were colonized by a diverse community of fungal endophytes in newly emerged tissue, but diversity decreased throughout the season leading to the preponderance of a single fungus in later sampling months. This previously undescribed endophyte appears to abundantly colonize the host to the detriment of other microfungi. Competition assays on a variety of media types failed to demonstrate that the dominant fungus was competitive against other fungi isolated from the same hosts, and inoculation onto sterile fern gametophytes did not alter growth compared to sterile controls, suggesting its effects are not antagonistic. The presence of this endophyte in the fern population probably demonstrates a case of repeated colonization driving competitive exclusion of other fungal community members.

Brain insulin resistance and cognitive function: influence of exercise.

Exercise has systemic health benefits in people, in part, through improving whole body insulin sensitivity. The brain is an insulin-sensitive organ that is often underdiscussed relative to skeletal muscle, liver, and adipose tissue. Although brain insulin action may have only subtle impacts on peripheral regulation of systemic glucose homeostasis, it is important for weight regulation as well as mental health. In fact, brain insulin signaling is also involved in processes that support healthy cognition. Furthermore, brain insulin resistance has been associated with age-related declines in memory and executive function as well as Alzheimer's disease pathology. Herein, we provide an overview of brain insulin sensitivity in relation to cognitive function from animal and human studies, with particular emphasis placed on the impact exercise may have on brain insulin sensitivity. Mechanisms discussed include mitochondrial function, brain growth factors, and neurogenesis, which collectively help combat obesity-related metabolic disease and Alzheimer's dementia.

Fic-mediated AMPylation tempers the unfolded protein response during physiological stress.

The proper balance of synthesis, folding, modification, and degradation of proteins, also known as protein homeostasis, is vital to cellular health and function. The unfolded protein response (UPR) is activated when the mechanisms maintaining protein homeostasis in the endoplasmic reticulum become overwhelmed. However, prolonged or strong UPR responses can result in elevated inflammation and cellular damage. Previously, we discovered that the enzyme filamentation induced by cyclic-AMP (Fic) can modulate the UPR response via posttranslational modification of binding immunoglobulin protein (BiP) by AMPylation during homeostasis and deAMPylation during stress. Loss of fic in Drosophila leads to vision defects and altered UPR activation in the fly eye. To investigate the importance of Fic-mediated AMPylation in a mammalian system, we generated a conditional null allele of Fic in mice and characterized the effect of Fic loss on the exocrine pancreas. Compared to controls, Fic-/- mice exhibit elevated serum markers for pancreatic dysfunction and display enhanced UPR signaling in the exocrine pancreas in response to physiological and pharmacological stress. In addition, both fic-/- flies and Fic-/- mice show reduced capacity to recover from damage by stress that triggers the UPR. These findings show that Fic-mediated AMPylation acts as a molecular rheostat that is required to temper the UPR response in the mammalian pancreas during physiological stress. Based on these findings, we propose that repeated physiological stress in differentiated tissues requires this rheostat for tissue resilience and continued function over the lifetime of an animal.

Insulin infusion decreases medium-sized extracellular vesicles in adults with metabolic syndrome.

Elevated extracellular vesicles (EVs) are associated with glucose dysmetabolism. However, the effects of insulin on EVs and subsequent relationships with insulin sensitivity, substrate oxidation, and inflammation are unknown. We tested the hypothesis that insulin would lower EVs and relate to insulin action. Fifty-one sedentary adults (54.8 ± 1.0 yr; V̇o2peak : 22.1 ± 0.6 mL/kg/min) with metabolic syndrome (MetS) and obesity (36.4 ± 0.65 kg/m2) underwent a 2-h euglycemic-hyperinsulinemic clamp (5 mmol/L; 40 mU/m2/min). Count and size (medium: 200-624 nm; larger: 625-1,000 nm) for total particle count, endothelial- (CD105+), leukocyte- (CD45+), platelet- (CD41+), and tetraspanin- (TX+: CD9/CD81/CD63), as well as platelet endothelial cell adhesion molecule- (CD31+) derived EVs were determined before and following the clamp using Full Spectrum Profiling (FSPM). Size and MESF (molecules of equivalent soluble fluorochrome) data were generated using FCMPASS Software. Fat and carbohydrate oxidation, in addition to high-sensitivity c-reactive protein (hsCRP), were measured to understand insulin effects and associations between EVs, metabolic flexibility, and inflammation. Despite low metabolic insulin sensitivity (M-Value = 2.56 ± 0.17 mg/kg/min), insulin increased carbohydrate (P = 0.015) and decreased fat oxidation (P = 0.048) and hsCRP (P = 0.016) compared with fasting. Insulin also decreased total particle count (P < 0.001), attributable to decreased medium-sized CD105+ (P = 0.052) and CD45+ EVs (P < 0.001). Elevated fasting insulin was associated with reduced insulin-stimulated changes in all EVs phenotypes (P < 0.001). Interestingly, fasting EVs were associated with increased fasting carbohydrate oxidation (all P < 0.05). These findings suggest that insulin decreases medium-sized EVs in conjunction with metabolic flexibility under euglycemic conditions in adults with MetS. More research is needed to determine how therapies alter EV phenotype/size and consequent cardiometabolic risk.NEW & NOTEWORTHY This study is one of the first to investigate the effects of insulin on medium and larger extracellular vesicles (EVs) in relation to metabolic insulin sensitivity and fuel use in adults with metabolic syndrome. Our data suggest that insulin infusion decreases the concentration of total particle counts, mainly due to reductions in medium-sized EVs. Furthermore, EVs, predominantly medium-sized, are inversely associated with metabolic flexibility.

Insulin Sensitivity and Metabolic Flexibility Parallel Plasma TCA Levels in Early Chronotype With Metabolic Syndrome.

CONTEXT: People characterized as late chronotype have elevated type 2 diabetes and cardiovascular disease risk compared to early chronotype. It is unclear how chronotype is associated with insulin sensitivity, metabolic flexibility, or plasma TCA cycle intermediates concentration, amino acids (AA), and/or beta-oxidation. OBJECTIVE: This study examined these metabolic associations with chronotype. METHODS: The Morningness-Eveningness Questionnaire (MEQ) was used to classify adults with metabolic syndrome (ATP III criteria) as either early (n = 15 [13F], MEQ = 64.7 ±â€…1.4) or late (n = 19 [16F], MEQ = 45.5 ±â€…1.3) chronotype. Fasting bloods determined hepatic (HOMA-IR) and adipose insulin resistance (Adipose-IR) while a 120-minute euglycemic clamp (40 mU/m2/min, 5 mmoL/L) was performed to test peripheral insulin sensitivity (glucose infusion rate). Carbohydrate (CHOOX) and fat oxidation (FOX), as well as nonoxidative glucose disposal (NOGD), were also estimated (indirect calorimetry). Plasma tricarboxylic acid cycle (TCA) intermediates, AA, and acyl-carnitines were measured along with VO2max and body composition (DXA). RESULTS: There were no statistical differences in age, BMI, fat-free mass, VO2max, or ATP III criteria between groups. Early chronotype, however, had higher peripheral insulin sensitivity (P = 0.009) and lower HOMA-IR (P = 0.02) and Adipose-IR (P = 0.05) compared with late chronotype. Further, early chronotype had higher NOGD (P = 0.008) and greater insulin-stimulated CHOOX (P = 0.02). While fasting lactate (P = 0.01), TCA intermediates (isocitrate, α-ketoglutarate, succinate, fumarate, malate; all P ≤ 0.04) and some AA (proline, isoleucine; P = 0.003-0.05) were lower in early chronotype, other AA (threonine, histidine, arginine; all P ≤ 0.05) and most acyl-carnitines were higher (P ≤ 0.05) compared with late chronotype. CONCLUSION: Greater insulin sensitivity and metabolic flexibility relates to plasma TCA concentration in early chronotype.

Role of Blood Pressure Responses to Exercise and Vascular Insulin Sensitivity with Nocturnal Blood Pressure Dipping in Metabolic Syndrome.

INTRODUCTION: Nocturnal systolic blood pressure (SBP) dipping is independently related to cardiovascular disease risk, but it is unclear if vascular insulin sensitivity associates with SBP dipping in patients with metabolic syndrome (MetS). METHODS: Eighteen adults with MetS (ATP III criteria 3.3 ± 0.6; 53.2 ± 6.5 years; body mass index 35.8 ± 4.5 kg/m2) were categorized as "dippers" (≥10% change in SBP; n = 4 F/3 M) or "non-dippers" (<10%; n = 9 F/2 M). Twenty-four-hour ambulatory blood pressure was recorded to assess SBP dipping. A euglycemic-hyperinsulinemic clamp (40 mU/m2/min, 90 mg/dL) with ultrasound (flow mediated dilation) was performed to test vascular insulin sensitivity. A graded, incremental exercise test was conducted to estimate sympathetic activity. Heart rate (HR) recovery after exercise was then used to determine parasympathetic activity. Metabolic panels and body composition (DXA) were also tested. RESULTS: Dippers had greater drops in SBP (16.63 ± 5.2 vs. 1.83 ± 5.6%, p < 0.01) and experienced an attenuated rise in both SBPslope (4.7 ± 2.3 vs. 7.2 ± 2.5 mm Hg/min, p = 0.05) and HRslope to the incremental exercise test compared to non-dippers (6.5 ± 0.9 vs. 8.2 ± 1.7 bpm/min, p = 0.03). SBP dipping correlated with higher insulin-stimulated flow-mediated dilation (r = 0.52, p = 0.03), although the relationship was no longer significant after covarying for HRslope (r = 0.42, p = 0.09). CONCLUSION: Attenuated rises in blood pressure and HR to exercise appear to play a larger role than vascular insulin sensitivity in SBP dipping in adults with MetS.

Calcium flux control by Pacs1-Wdr37 promotes lymphocyte quiescence and lymphoproliferative diseases.

Endoplasmic reticulum (ER) calcium (Ca2+ ) stores are critical to proteostasis, intracellular signaling, and cellular bioenergetics. Through forward genetic screening in mice, we identified two members of a new complex, Pacs1 and Wdr37, which are required for normal ER Ca2+ handling in lymphocytes. Deletion of Pacs1 or Wdr37 caused peripheral lymphopenia that was linked to blunted Ca2+ release from the ER after antigen receptor stimulation. Pacs1-deficient cells showed diminished inositol triphosphate receptor expression together with increased ER and oxidative stress. Mature Pacs1-/- B cells proliferated and died in vivo under lymphocyte replete conditions, indicating spontaneous loss of cellular quiescence. Disruption of Pacs1-Wdr37 did not diminish adaptive immune responses, but potently suppressed lymphoproliferative disease models by forcing loss of quiescence. Thus, Pacs1-Wdr37 plays a critical role in stabilizing lymphocyte populations through ER Ca2+ handling and presents a new target for lymphoproliferative disease therapy.

Metformin May Contribute to Inter-individual Variability for Glycemic Responses to Exercise.

Metformin and exercise independently improve glycemic control. Metformin traditionally is considered to reduce hepatic glucose production, while exercise training is thought to stimulate skeletal muscle glucose disposal. Collectively, combining treatments would lead to the anticipation for additive glucose regulatory effects. Herein, we discuss recent literature suggesting that metformin may inhibit, enhance or have no effect on exercise mediated benefits toward glucose regulation, with particular emphasis on insulin sensitivity. Importantly, we address issues surrounding the impact of metformin on exercise induced glycemic benefit across multiple insulin sensitive tissues (e.g., skeletal muscle, liver, adipose, vasculature, and the brain) in effort to illuminate potential sources of inter-individual glycemic variation. Therefore, the review identifies gaps in knowledge that require attention in order to optimize medical approaches that improve care of people with elevated blood glucose levels and are at risk of cardiovascular disease.

No island-effect on glucocorticoid levels for a rodent from a near-shore archipelago.

Island rodents are often larger and live at higher population densities than their mainland counterparts, characteristics that have been referred to as "island syndrome". Island syndrome has been well studied, but few studies have tested for island-mainland differences in stress physiology. We evaluated island syndrome within the context of stress physiology of white-footed mice (Peromyscus leucopus) captured from 11 islands and five mainland sites in Thousand Islands National Park, Ontario, Canada. Stress physiology was evaluated by quantifying corticosterone (a stress biomarker), the primary glucocorticoid in mice, from hair and its related metabolites from fecal samples. White-footed mice captured in this near-shore archipelago did not display characteristics of island syndrome, nor differences in levels of hair corticosterone or fecal corticosterone metabolites compared with mainland mice. We suggest that island white-footed mice experience similar degrees of stress in the Thousand Islands compared with the mainland. Although we did not find evidence of island syndrome or differences in glucocorticoid levels, we identified relationships between internal (sex, body mass) and external (season) factors and our hormonal indices of stress in white-footed mice.

Risk factors that predict severe injuries in university rugby sevens players.

OBJECTIVES: To investigate injury incidence and the influence of physical fitness parameters on the risk of severe injuries in players on rugby sevens university teams. DESIGN: Prospective cohort study. METHODS: Rugby players from three universities (N=104; 90M:14F; 20.6±1.9years) were recruited before the beginning of the season. Players underwent pre-season assessments of power, strength, speed, agility, endurance, stability, and flexibility. Throughout the season, rugby-related injury and exposure data were collected. Potential predictor variables were analyzed using Cox proportional regression model to identify risk factors associated with severe injuries (time loss>28days). RESULTS: Thirty-one injuries occurred during the rugby season. The match and training injury incidence rates were 59.3 injuries and 3.3 injuries per 1000 player-hours, respectively. Lower limb injuries were most common and most severe. The ankle joint was the most prevalent site of injury, and ligamentous injury was most common (48.4%). Nine severe injuries were sustained resulting in an average time loss of 51.3±14.6days. Female (hazard ratio [HR]=8.35; 95% confidence intervals [CI]=2.01-34.8), slower (HR=3.51; 95% CI=1.17-10.5), and less agile (HR=2.22; 95% CI=1.26-3.92) players as well as those with hip flexors tightness (HR=1.12; 95% CI=1.00-1.25) were at significantly greater risk for sustaining severe injuries. CONCLUSIONS: Limited studies are available on risk factors associated with amateur rugby players in the Sevens version. The development of gender-specific injury prevention measures that emphasize speed and agility training, and improve hip flexor extensibility may be important to reduce the risk of severe injuries.

Testing the nutritional-limitation, predator-avoidance, and storm-avoidance hypotheses for restricted sea otter habitat use in the Aleutian Islands, Alaska.

Sea otters (Enhydra lutris) inhabiting the Aleutian Islands have stabilized at low abundance levels following a decline and currently exhibit restricted habitat-utilization patterns. Possible explanations for restricted habitat use by sea otters can be classified into two fundamentally different processes, bottom-up and top-down forcing. Bottom-up hypotheses argue that changes in the availability or nutritional quality of prey resources have led to the selective use of habitats that support the highest quality prey. In contrast, top-down hypotheses argue that increases in predation pressure from killer whales have led to the selective use of habitats that provide the most effective refuge from killer whale predation. A third hypothesis suggests that current restricted habitat use is based on a need for protection from storms. We tested all three hypotheses for restricted habitat use by comparing currently used and historically used sea otter foraging locations for: (1) prey availability and quality, (2) structural habitat complexity, and (3) exposure to prevailing storms. Our findings suggest that current use is based on physical habitat complexity and not on prey availability, prey quality, or protection from storms, providing further evidence for killer whale predation as a cause for restricted sea otter habitat use in the Aleutian Islands.

Comparative functional genomics of adaptation to muscular disuse in hibernating mammals.

Hibernation is an energy-saving adaptation that involves a profound suppression of physical activity that can continue for 6-8 months in highly seasonal environments. While immobility and disuse generate muscle loss in most mammalian species, in contrast, hibernating bears and ground squirrels demonstrate limited muscle atrophy over the prolonged periods of physical inactivity during winter, suggesting that hibernating mammals have adaptive mechanisms to prevent disuse muscle atrophy. To identify common transcriptional programmes that underlie molecular mechanisms preventing muscle loss, we conducted a large-scale gene expression screen in hind limb muscles comparing hibernating and summer-active black bears and arctic ground squirrels using custom 9600 probe cDNA microarrays. A molecular pathway analysis showed an elevated proportion of overexpressed genes involved in all stages of protein biosynthesis and ribosome biogenesis in muscle of both species during torpor of hibernation that suggests induction of translation at different hibernation states. The induction of protein biosynthesis probably contributes to attenuation of disuse muscle atrophy through the prolonged periods of immobility of hibernation. The lack of directional changes in genes of protein catabolic pathways does not support the importance of metabolic suppression for preserving muscle mass during winter. Coordinated reduction in multiple genes involved in oxidation-reduction and glucose metabolism detected in both species is consistent with metabolic suppression and lower energy demand in skeletal muscle during inactivity of hibernation.

Preservation of bone mass and structure in hibernating black bears (Ursus americanus) through elevated expression of anabolic genes.

Physical inactivity reduces mechanical load on the skeleton, which leads to losses of bone mass and strength in non-hibernating mammalian species. Although bears are largely inactive during hibernation, they show no loss in bone mass and strength. To obtain insight into molecular mechanisms preventing disuse bone loss, we conducted a large-scale screen of transcriptional changes in trabecular bone comparing winter hibernating and summer non-hibernating black bears using a custom 12,800 probe cDNA microarray. A total of 241 genes were differentially expressed (P < 0.01 and fold change >1.4) in the ilium bone of bears between winter and summer. The Gene Ontology and Gene Set Enrichment Analysis showed an elevated proportion in hibernating bears of overexpressed genes in six functional sets of genes involved in anabolic processes of tissue morphogenesis and development including skeletal development, cartilage development, and bone biosynthesis. Apoptosis genes demonstrated a tendency for downregulation during hibernation. No coordinated directional changes were detected for genes involved in bone resorption, although some genes responsible for osteoclast formation and differentiation (Ostf1, Rab9a, and c-Fos) were significantly underexpressed in bone of hibernating bears. Elevated expression of multiple anabolic genes without induction of bone resorption genes, and the down regulation of apoptosis-related genes, likely contribute to the adaptive mechanism that preserves bone mass and structure through prolonged periods of immobility during hibernation.

Chromomycin SA analogs from a marine-derived Streptomyces sp.

Two chromomycin SA analogs, chromomycin SA(3) and chromomycin SA(2), along with deacetylchromomycin A(3) and five previously reported chromomycin analogs were isolated from a marine-derived Streptomyces sp. The structures of the new compounds were determined by spectroscopic methods including 1D and 2D NMR techniques, HRMS and chemical methods. Chromomycin SA(3) and chromomycin SA(2) are the first naturally occuring chromomycin analogs with truncated side-chains. Biological evaluation of chromomycin analogs for cytotoxicity against two non-small cell lung cancer (NSCLC) cell-lines, A549 and HCC44, demonstrated a decrease in cytotoxicity for the truncated sides chain chromomycin analogs.

Modulation of gene expression in heart and liver of hibernating black bears (Ursus americanus).

BACKGROUND: Hibernation is an adaptive strategy to survive in highly seasonal or unpredictable environments. The molecular and genetic basis of hibernation physiology in mammals has only recently been studied using large scale genomic approaches. We analyzed gene expression in the American black bear, Ursus americanus, using a custom 12,800 cDNA probe microarray to detect differences in expression that occur in heart and liver during winter hibernation in comparison to summer active animals. RESULTS: We identified 245 genes in heart and 319 genes in liver that were differentially expressed between winter and summer. The expression of 24 genes was significantly elevated during hibernation in both heart and liver. These genes are mostly involved in lipid catabolism and protein biosynthesis and include RNA binding protein motif 3 (Rbm3), which enhances protein synthesis at mildly hypothermic temperatures. Elevated expression of protein biosynthesis genes suggests induction of translation that may be related to adaptive mechanisms reducing cardiac and muscle atrophies over extended periods of low metabolism and immobility during hibernation in bears. Coordinated reduction of transcription of genes involved in amino acid catabolism suggests redirection of amino acids from catabolic pathways to protein biosynthesis. We identify common for black bears and small mammalian hibernators transcriptional changes in the liver that include induction of genes responsible for fatty acid ß oxidation and carbohydrate synthesis and depression of genes involved in lipid biosynthesis, carbohydrate catabolism, cellular respiration and detoxification pathways. CONCLUSIONS: Our findings show that modulation of gene expression during winter hibernation represents molecular mechanism of adaptation to extreme environments.