Impact assessment of metabolite instability in the development and validation of LC-MS/MS bioanalytical assays for measurement of rosuvastatin in human plasma and urine samples.

During bioanalytical assay development and validation, maintaining the stability of the parent drug and metabolites of interest is critical. While stability of the parent drug has been thoroughly investigated, the stability of unanalyzed metabolites is often overlooked. When an unstable metabolite is known or suspected to interfere with measurement of the parent drug or other metabolites of interest through back-conversion or other routes, additional tests with these unstable metabolites should be conducted. Here, the development and validation of two assays for quantification of rosuvastatin, one in human plasma and one in human urine, was reported. To this end, additional sets of quality control samples were added during assay validation to ensure the reliability of the assays. Acid treatment of samples is shown to be necessary for rosuvastatin quantification. In this regard, stability issues caused by the metabolite, rosuvastatin lactone, may have been overlooked if assay development and validation had only considered the parent drug, rosuvastatin. These assays represent a case study for how to develop and validate assays with unstable metabolites. Taken together, unstable metabolites should be included in all applicable stability tests.

Promoter Length Affects the Initiation of T7 RNA Polymerase In Vitro: New Insights into Promoter/Polymerase Co-evolution.

Polymerases are integral factors of gene expression and are essential for the maintenance and transmission of genetic information. RNA polymerases (RNAPs) differ from other polymerases in that they can bind promoter sequences and initiate transcription de novo and this promoter recognition requires the presence of specific DNA binding domains in the polymerase. Bacteriophage T7 RNA polymerase (T7RNAP) is the prototype for single subunit RNA polymerases which include bacteriophage and mitochondrial RNAPs, and the structure and mechanistic aspects of transcription by T7 RNAP are well characterized. Here, we describe experiments to determine whether the prototype T7 RNAP is able to recognize and initiate at truncated promoters similar to mitochondrial promoters. Using an in vitro oligonucleotide transcriptional system, we have assayed transcription initiation activity by T7 RNAP. These assays have not only defined the limits of conventional de novo initiation on truncated promoters, but have identified novel activities of initiation of RNA synthesis. We propose that these novel activities may be vestigial activities surviving from the transition of single subunit polymerase initiation using primers to de novo initiation using promoters.

Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform.

The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-L-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l(-1)) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.

Microbiota associated with tubes of Escarpia sp. from cold seeps in the southwestern Atlantic Ocean constitutes a community distinct from that of surrounding marine sediment and water.

As the depth increases and the light fades in oceanic cold seeps, a variety of chemosynthetic-based benthic communities arise. Previous assessments reported polychaete annelids belonging to the family Siboglinidae as part of the fauna at cold seeps, with the 'Vestimentifera' clade containing specialists that depend on microbial chemosynthetic endosymbionts for nutrition. Little information exists concerning the microbiota of the external portion of the vestimentiferan trunk wall. We employed 16S rDNA-based metabarcoding to describe the external microbiota of the chitin tubes from the vestimentiferan Escarpia collected from a chemosynthetic community in a cold seep area at the southwestern Atlantic Ocean. The most abundant operational taxonomic unit (OTU) belonged to the family Pirellulaceae (phylum Planctomycetes), and the second most abundant OTU belonged to the order Methylococcales (phylum Proteobacteria), composing an average of 21.1 and 15.4% of the total reads on tubes, respectively. These frequencies contrasted with those from the surrounding environment (sediment and water), where they represent no more than 0.1% of the total reads each. Moreover, some taxa with lower abundances were detected only in Escarpia tube walls. These data constitute on the first report of an epibiont microbial community found in close association with external surface of a cold-seep metazoan, Escarpia sp., from a chemosynthetic community in the southwestern Atlantic Ocean.

Detection of chemical warfare agent simulants and hydrolysis products in biological samples by paper spray mass spectrometry.

Paper spray ionization coupled to a high resolution tandem mass spectrometer (a quadrupole orbitrap) was used to identify and quantitate chemical warfare agent (CWA) simulants and their hydrolysis products in blood and urine. Three CWA simulants, dimethyl methylphosphonate (DMMP), trimethyl phosphate (TMP), and diisopropyl methylphosphonate (DIMP), and their isotopically labeled standards were analyzed in human whole blood and urine. Calibration curves were generated and tested with continuing calibration verification standards. Limits of detection for these three compounds were in the low ng mL-1 range for the direct analysis of both blood and urine samples. Five CWA hydrolysis products, ethyl methylphosphonic acid (EMPA), isopropyl methylphosphonic acid (IMPA), isobutyl methylphosphonic acid (iBuMPA), cyclohexyl methylphosphonic acid (CHMPA), and pinacolyl methylphosphonic acid (PinMPA), were also analyzed. Calibration curves were generated in both positive and negative ion modes. Limits of detection in the negative ion mode ranged from 0.36 ng mL-1 to 1.25 ng mL-1 in both blood and urine for the hydrolysis products. These levels were well below those found in victims of the Tokyo subway attack of 2 to 135 ng mL-1. Improved stability and robustness of the paper spray technique in the negative ion mode was achieved by the addition of chlorinated solvents. These applications demonstrate that paper spray mass spectrometry (PS-MS) can be used for rapid, sample preparation-free detection of chemical warfare agents and their hydrolysis products at physiologically relevant concentrations in biological samples.

Younger rats are more susceptible to the lethal effects of sarin than adult rats: 24 h LC50 for whole-body (10 and 60 min) exposures.

Chemical warfare nerve agents (CWNA) inhibit acetylcholinesterase and are among the most lethal chemicals known to man. Children are predicted to be vulnerable to CWNA exposure because of their smaller body masses, higher ventilation rates and immature central nervous systems. While a handful of studies on the effects of CWNA in younger animals have been published, exposure routes relevant to battlefield or terrorist situations (i.e. inhalation for sarin) were not used. Thus, we estimated the 24 h LC50 for whole-body (10 and 60 min) exposure to sarin using a stagewise, adaptive dose design. Specifically, male and female Sprague-Dawley rats were exposed to a range of sarin concentrations (6.2-44.0 or 1.6-12.5 mg/m³) for either 10 or 60 min, respectively, at six different times during their development (postnatal day [PND] 7, 14, 21, 28, 42 and 70). For male and female rats, the lowest LC50 values were observed for PND 14 and the highest LC50 values for PND 28. Sex differences were observed only for PND 42 for the 10 min exposures and PND 21 and 70 for the 60 min exposures. Thus, younger rats (PND 14) were more susceptible than older rats (PND 70) to the lethal effects of whole-body exposure to sarin, while adolescent (PND 28) rats were the least susceptible and sex differences were minimal. These results underscore the importance of controlling for the age of the animal in research on the toxic effects associated with CWNA exposure.

Nonclinical Profile of BLZ-100, a Tumor-Targeting Fluorescent Imaging Agent.

BLZ-100 is a single intravenous use, fluorescent imaging agent that labels tumor tissue to enable more complete and precise surgical resection. It is composed of a chlorotoxin peptide covalently bound to the near-infrared fluorophore indocyanine green. BLZ-100 is in clinical development for intraoperative visualization of human tumors. The nonclinical safety and pharmacokinetic (PK) profile of BLZ-100 was evaluated in mice, rats, canines, and nonhuman primates (NHP). Single bolus intravenous administration of BLZ-100 was well tolerated, and no adverse changes were observed in cardiovascular safety pharmacology, PK, and toxicology studies in rats and NHP. The single-dose no-observed-adverse-effect-levels (NOAELs) were 7 mg (28 mg/kg) in rats and 60 mg (20 mg/kg) in NHP, corresponding to peak concentration values of 89 400 and 436 000 ng/mL and area-under-the-curve exposure values of 130 000 and 1 240 000 h·ng/mL, respectively. Based on a human imaging dose of 3 mg, dose safety margins are >100 for rat and monkey. BLZ-100 produced hypersensitivity reactions in canine imaging studies (lethargy, pruritus, swollen muzzle, etc). The severity of the reactions was not dose related. In a follow-up study in dogs, plasma histamine concentrations were increased 5 to 60 minutes after BLZ-100 injection; this coincided with signs of hypersensitivity, supporting the conclusion that the reactions were histamine based. Hypersensitivity reactions were not observed in other species or in BLZ-100 human clinical studies conducted to date. The combined imaging, safety pharmacology, PK, and toxicology studies contributed to an extensive initial nonclinical profile for BLZ-100, supporting first-in-human clinical trials.

Cocaine occupancy of sigma1 receptors and dopamine transporters in mice.

Activation of sigma1 (σ1) receptors contributes to the behavioral and toxic effects of (-)-cocaine. We studied a key step, the ability of (-)-cocaine to occupy σ1 receptors in vivo, using CD-1(®) mice and the novel radioligand [(125) I]E-N-1-(3'-iodoallyl)-N'-4-(3",4"-dimethoxyphenethyl)-piperazine ([(125) I]E-IA-DM-PE-PIPZE). (-)-Cocaine displayed an ED50 of 68 µmol/kg for inhibition of specific radioligand binding in whole brain, with values between 73 and 80 µmol/kg for heart, lung, and spleen. For comparison, an ED50 of 26 µmol/kg for (-)-cocaine occupancy of striatal dopamine transporters (DAT) was determined by inhibition of [(125) I]3ß-(4-iodophenyl)tropan-2ß-carboxylic acid isopropyl ester ([(125) I]RTI-121) binding. A chief finding is the relatively small potency difference between (-)-cocaine occupancy of σ1 receptors and the DAT, although the DAT occupancy is likely underestimated. Interactions of (-)-cocaine with σ1 receptors were assessed further using [(125) I]E-IA-DM-PE-PIPZE for regional cerebral biodistribution studies and quantitative ex vivo autoradiography of brain sections. (-)-Cocaine binding to cerebral σ1 receptors proved directly proportional to the relative site densities known for the brain regions. Nonradioactive E-IA-DM-PE-PIPZE gave an ED50 of 0.23 µmol/kg for occupancy of cerebral σ1 receptors, and a 3.16 µmol/kg (i.p.) dose attenuated (-)-cocaine-induced locomotor hyperactivity by 30%. This effect did not reach statistical significance, but suggests that E-IA-DM-PE-PIPZE is a probable σ1 receptor antagonist. As groundwork for the in vivo studies, we used standard techniques in vitro to determine ligand affinities, site densities, and pharmacological profiles for the σ1 and σ2 receptors expressed in CD-1(®) mouse brain.

Relationship between cerebral sigma-1 receptor occupancy and attenuation of cocaine's motor stimulatory effects in mice by PD144418.

Psychostimulant effects of cocaine are mediated partly by agonist actions at sigma-1 (σ1) receptors. Selective σ1 receptor antagonists attenuate these effects and provide a potential avenue for pharmacotherapy. However, the selective and high affinity σ1 antagonist PD144418 (1,2,3,6-tetrahydro-5-[3-(4-methylphenyl)-5-isoxazolyl]-1-propylpyridine) has been reported not to inhibit cocaine-induced hyperactivity. To address this apparent paradox, we evaluated aspects of PD144418 binding in vitro, investigated σ1 receptor and dopamine transporter (DAT) occupancy in vivo, and re-examined effects on locomotor activity. PD144418 displayed high affinity for σ1 sites (Ki 0.46 nM) and 3596-fold selectivity over σ2 sites (Ki 1654 nM) in guinea pig brain membranes. No appreciable affinity was noted for serotonin and norepinephrine transporters (Ki >100 µM), and the DAT interaction was weak (Ki 9.0 µM). In vivo, PD144418 bound to central and peripheral σ1 sites in mouse, with an ED50 of 0.22 µmol/kg in whole brain. No DAT occupancy by PD144418 (10.0 µmol/kg) or possible metabolites were observed. At doses that did not affect basal locomotor activity, PD144418 (1, 3.16, and 10 µmol/kg) attenuated cocaine-induced hyperactivity in a dose-dependent manner in mice. There was good correlation (r(2) = 0.88) of hyperactivity reduction with increasing cerebral σ1 receptor occupancy. The behavioral ED50 of 0.79 µmol/kg corresponded to 80% occupancy. Significant σ1 receptor occupancy and the ability to mitigate cocaine's motor stimulatory effects were observed for 16 hours after a single 10.0 µmol/kg dose of PD144418.

A selective sigma-2 receptor ligand antagonizes cocaine-induced hyperlocomotion in mice.

Cocaine functions, in part, through agonist actions at sigma-1 (σ1 ) receptors, while roles played by sigma-2 (σ2 ) receptors are less established. Attempts to discriminate σ2 receptor-mediated effects of cocaine in locomotor hyperactivity assays have been hampered by the lack of potent and selective antagonists. Certain tetrahydroisoquinolinyl benzamides display high σ2 receptor affinity, and excellent selectivity for binding to σ2 over σ1 receptors. The behavioral properties of this structural class of σ ligands have not yet been investigated. The present study evaluated 5-bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl)]-2,3-dimethoxy-benzamide, 1, a ligand shown by others to bind preferentially to σ2 over σ1 receptors, as well as dopamine D2 and D3 sites. First, we determined binding to monoamine transporters and opioid receptors, and noted 57-fold selectivity for σ2 receptors over the serotonin transporter, and >800-fold selectivity for σ2 receptors over the other sites tested. We then examined 1 in locomotor activity studies using male CD-1® mice, and saw no alteration of basal activity at doses up to 31.6 µmol/kg. Cocaine produced a fivefold increase in locomotor activity, which was attenuated by 66% upon pretreatment of mice with 1 at 31.6 µmol/kg. In vivo radioligand binding studies also were performed, and showed no occupancy of σ1 receptors or the dopamine transporter by 1, or its possible metabolites, at the 31.6 µmol/kg dose. Thus, ligand 1 profiles behaviorally as a σ2 receptor-selective antagonist that is able to counteract cocaine's motor stimulatory effects.

Comparison of sarin and cyclosarin toxicity by subcutaneous, intravenous and inhalation exposure in Gottingen minipigs.

Sexually mature male and female Gottingen minipigs were exposed to various concentrations of GB and GF vapor via whole-body inhalation exposures or to liquid GB or GF via intravenous or subcutaneous injections. Vapor inhalation exposures were for 10, 60 or 180 min. Maximum likelihood estimation was used to calculate the median effect levels for severe effects (ECT50 and ED50) and lethality (LCT50 and LD50). Ordinal regression was used to model the concentration × time profile of the agent toxicity. Contrary to that predicted by Haber's rule, LCT50 values increased as the duration of the exposures increased for both nerve agents. The toxic load exponents (n) were calculated to be 1.38 and 1.28 for GB and GF vapor exposures, respectively. LCT50 values for 10-, 60- and 180-min exposures to vapor GB in male minipigs were 73, 106 and 182 mg min/m(3), respectively. LCT50 values for 10-, 60 - and 180-min exposures to vapor GB in female minipigs were 87, 127 and 174 mg min/m(3), respectively. LCT50 values for 10-, 60- and 180-min exposures to vapor GF in male minipigs were 218, 287 and 403 mg min/m(3), respectively. LCT50 values for 10-, 60- and 180-min exposures in female minipigs were 183, 282 and 365 mg min/m(3), respectively. For GB vapor exposures, there was a tenuous gender difference which did not exist for vapor GF exposures. Surprisingly, GF was 2-3 times less potent than GB via the inhalation route of exposure regardless of exposure duration. Additionally GF was found to be less potent than GB by intravenous and subcutaneous routes.

Genetic Diversity in the mtDNA of Physarum polycephalum.

The mtDNA of the myxomycete Physarum polycephalum can contain as many as 81 genes. These genes can be grouped in three different categories. The first category includes 46 genes that are classically found on the mtDNA of many organisms. However, 43 of these genes are cryptogenes that require a unique type of RNA editing (MICOTREM). A second category of gene is putative protein-coding genes represented by 26 significant open reading frames. However, these genes do not appear to be transcribed during the growth of the plasmodium and are currently unassigned since they do not have any apparent similarity to other classical mitochondrial protein-coding genes. The third category of gene is found in the mtDNA of some strains of P. polycephalum. These genes derive from a linear mitochondrial plasmid with nine significant, but unassigned, open reading frames which can integrate into the mitochondrial DNA by recombination. Here, we review the mechanism and evolution of the RNA editing necessary for cryptogene expression, discuss possible origins for the 26 unassigned open reading frames based on tentative identification of their protein product, and discuss the implications to mtDNA structure and replication of the integration of the linear mitochondrial plasmid.

Iron from Co-Encapsulation of Defatted Nannochloropsis Oceanica with Inulin Is Highly Bioavailable and Does Not Impact Wheat Flour Shelf Life or Sensorial Attributes.

Defatted green microalgae Nannochloropsis oceanica (DGM) is a rich source of bioavailable iron. However, its use in foods results in unacceptable color and taste development. Therefore, the purpose of this study was to investigate strategies to enhance the use of DGM in foods. DGM and inulin were encapsulated (EC) in an oil-in-water emulsion using high-pressure homogenization. To confirm iron bioavailability, C57BL/6 mice were fed an iron-deficient diet (ID) for 2 weeks. The mice were then fed one of the four diets: ID, ID + DGM (DGM), ID + EC (EC50 or EC100) for 4 weeks. To test the stability of DGM as an iron fortificant at two different fortification rates of 17.5 mg Fe/kg (50%) or 35 mg Fe/kg (100%), whole (DGM50/DGM100), encapsulated (EC50/EC100) and color-masked (CM50/CM100) DGM were added to wheat flour (WF) at two different temperatures: 20 °C and 45 °C and were examined for 30 days. Acceptability studies were conducted to determine sensory differences between rotis (Indian flat bread) prepared from WF/EC50/CM50/EC100. The mice consuming EC50/EC100 diets showed comparable iron status to DGM-fed mice, suggesting that encapsulation did not negatively impact iron bioavailability. Addition of EC to wheat flour resulted in the lowest Fe2+ oxidation and color change amongst treatments, when stored for 30 days. There were no differences in the overall liking and product acceptance of rotis amongst treatments at both day 0 and day 21 samples. Our results suggest that EC50 can be effectively used as an iron fortificant in WF to deliver highly bioavailable iron without experiencing any stability or sensory defects, at least until 30 days of storage.

Validation of LC-MS/MS methods for quantitative analysis of kynurenine pathway metabolites in human plasma and cerebrospinal fluid.

Background: Dysregulation of the kynurenine metabolic pathway has been reported in several neurological conditions. Methods & results: Sensitive and selective LC-MS/MS methods have been validated for six kynurenine pathway metabolites in human cerebrospinal fluid and plasma. For each matrix, we validated three methods - one for the simultaneous determination of kynurenine, kynurenic acid, anthranilic acid and 3-hydroxy-kynurenine (four-analyte assay), one for quinolinic acid and one for tryptophan - using stable-isotopically labeled internal standards. The dynamic range and quantitation limits were based on endogenous concentrations for each analyte. Conclusion: The use of validated methods for kynurenine pathway metabolites in human cerebrospinal fluid and plasma will provide definitive information in neurological diseases.

Informed surgical consent for a mesh/graft-augmented vaginal repair of pelvic organ prolapse. Consensus of the 2nd IUGA Grafts Roundtable: optimizing safety and appropriateness of graft use in transvaginal pelvic reconstructive surgery.

INTRODUCTION AND HYPOTHESIS: Complex issues surround informed surgical consent for pelvic reconstructive surgery. METHODS: Vaginally placed mesh/grafts are used with the intent to increase durability of the repair but potentially introduce unique complications, offering new challenges in informed surgical consent counseling. RESULTS: Informed consent is a process that takes place throughout the entire consultation with the patient. A written document often accompanies that process. This paper outlines necessary components of informed surgical consent and the theory behind each component. CONCLUSIONS: We explore elements that should be included in the consent process with regard to expected benefits, alternatives, and material risks that are specific to the use of a mesh/graft-augmented vaginal repair of prolapse. Included is an appendix that may serve as a template for the creation of a surgeon's own written informed consent document.

Preclinical safety, pharmacokinetics, and pharmacodynamics of recombinant human interleukin-21 in cynomolgus macaques (Macaca fascicularis).

Interleukin-21 (IL-21), a pleiotropic immunostimulatory type I cytokine, has anticancer effects in animal models. Preclinical studies designed to assess the safety of recombinant human IL-21 (rIL-21) for use in phase I oncology studies are described. The rIL-21 (≤3.0 mg/kg per dose) was given intravenously to cynomolgus monkeys (Macaca fascicularis) once daily for 5 days, followed by 9 nondosing days (1 cycle) for ≤4 cycles. The rIL-21 pharmacokinetics was dose-dependent. Accumulation was not observed after repeated dosing, consistent with the short elimination half-life (t (1/2,λz); 0.4-0.8 hours). Safety findings included cyclical anemia and thrombocytopenia, clinical pathology changes consistent with acute-phase response, leukocyte infiltrates in hepatic sinusoids, and sporadic serum transaminase elevations (typically <3 times upper-limit of normal); all were reversible upon cessation of treatment. Decreased pharmacodynamic responses with time corresponded to the development of anti-rIL-21 antibodies; effects varied among individuals and were dose-dependent. These studies demonstrated rIL-21 to be generally well-tolerated when administered to cynomolgus monkeys, and all adverse effects were reversible upon treatment cessation. However, development of anti-rIL-21 antibodies may limit the use of this species in long-term studies.

A first-in-human study of BLZ-100 (tozuleristide) demonstrates tolerability and safety in skin cancer patients.

BLZ-100 (tozuleristide) is an intraoperative fluorescent imaging agent that selectively detects malignant tissue and can be used in real time to guide tumor resection. The purpose of this study was to assess the safety, tolerability, and pharmacokinetics of BLZ-100 and to explore the pharmacodynamics of fluorescence imaging of skin tumors. In this first-in-human study, BLZ-100 was administered intravenously to 21 adult patients 2 days before excising known or suspected skin cancers. Doses were 1, 3, 6, 12, and 18 mg, with 3-6 patients/cohort. Fluorescence imaging was conducted before and up to 48 h after dosing. BLZ-100 was well tolerated. There were no serious adverse events, deaths, or discontinuations due to adverse events, and no maximum tolerated dose (MTD) was identified. Headache (n = 2) and nausea (n = 2) were the only BLZ-100 treatment-related adverse events reported for >1 patient. Median time to maximal serum concentration was <0.5 h. Exposure based on maximal serum concentrations increased in a greater than dose-proportional manner. For intermediate dose-levels (3-12 mg), 4 of 5 basal cell carcinomas and 4 of 4 melanomas were considered positive for BLZ-100 fluorescence. BLZ-100 was well tolerated at all dose levels tested and these results support further clinical testing of this imaging agent in surgical oncology settings. Clinicaltrials.gov: NCT02097875.

RNA-sequencing and behavioral testing reveals inherited physical inactivity co-selects for anxiogenic behavior without altering depressive-like behavior in Wistar rats.

Physical inactivity is positively associated with anxiety and depression. Considering physical inactivity, anxiety, and depression each have a genetic basis for inheritance, our lab used artificial selectively bred low-voluntary running (LVR) and wild type (WT) female Wistar rats to test if physical inactivity genes selected over multiple generations would lead to an anxiety or depressive-like phenotype. We performed next generation RNA sequencing and immunoblotting on the dentate gyrus to reveal key biological functions from heritable physical inactivity. LVR rats did not display depressive-like behavior. However, LVR rats did display anxiogenic behavior with gene networks associated with reduced neuronal development, proliferation, and function compared to WT counterparts. Additionally, immunoblotting revealed LVR deficits in neuronal development and function. To our knowledge, this is the first study to show that by selectively breeding for physical inactivity genes, anxiety-like genes were co-selected. The study also reveals molecular insights to the genetic influences that physical inactivity has on anxiety-like behavior.

Supplemental dietary inulin of variable chain lengths alters intestinal bacterial populations in young pigs.

Previously, we showed that supplementation of diets with short-chain inulin (P95), long-chain inulin (HP), and a 50:50 mixture of both (Synergy 1) improved body iron status and altered expression of the genes involved in iron homeostasis and inflammation in young pigs. However, the effects of these 3 types of inulin on intestinal bacteria remain unknown. Applying terminal restriction fragment length polymorphism analysis, we determined the abundances of luminal and adherent bacterial populations from 6 segments of the small and large intestines of pigs (n = 4 for each group) fed an iron-deficient basal diet (BD) or the BD supplemented with 4% of P95, Synergy 1, or HP for 5 wk. Compared with BD, all 3 types of inulin enhanced (P < 0.05) the abundance of beneficial bifidobacteria and lactobacilli in the microbiota adherent to intestinal mucus of various gut segments of pigs. These changes were seen as proximal as in the jejunum with P95 but did not appear until the distal ileum or cecum with HP. Similar effects of inulin on bacterial populations in the lumen contents were found. Meanwhile, all 3 types of inulin suppressed the less desirable bacteria Clostridium spp. and members of the Enterobacteriaceae in the lumen and mucosa of various gut segments. Our findings suggest that the ability of dietary inulin to alter intestinal bacterial populations may partially account for its iron bioavailability-promoting effect and possibly other health benefits.

Are inland wolf-ungulate systems influenced by marine subsidies of Pacific salmon?

Wolves (Canis lupus) in North America are considered obligate predators of ungulates with other food resources playing little role in wolf population dynamics or wolf prey relations. However, spawning Pacific salmon (Oncorhyncus spp.) are common throughout wolf range in northwestern North America and may provide a marine subsidy affecting inland wolf-ungulate food webs far from the coast. We conducted stable-isotope analyses for nitrogen and carbon to evaluate the contribution of salmon to diets of wolves in Denali National Park and Preserve, 1200 river-km from tidewater in interior Alaska, USA. We analyzed bone collagen from 73 wolves equipped with radio collars during 1986-2002 and evaluated estimates of salmon in their diets relative to the availability of salmon and ungulates within their home ranges. We compared wolf densities and ungulate:wolf ratios among regions with differing salmon and ungulate availability to assess subsidizing effects of salmon on these wolf-ungulate systems. Wolves in the northwestern flats of the study area had access to spawning salmon but low ungulate availability and consumed more salmon (17% +/- 7% [mean +/- SD]) than in upland regions, where ungulates were sixfold more abundant and wolves did or did not have salmon spawning areas within their home ranges (8% +/- 6% and 3% +/- 3%, respectively). Wolves were only 17% less abundant on the northwestern flats compared to the remainder of the study area, even though ungulate densities were 78% lower. We estimated that biomass from fall runs of chum (O. keta) and coho (O. kisutch) salmon on the northwestern flats was comparable to the ungulate biomass there, and the contribution of salmon to wolf diets was similar to estimates reported for coastal wolves in southeast Alaska. Given the ubiquitous consumption of salmon by wolves on the northwestern flats and the abundance of salmon there, we conclude that wolf numbers in this region were enhanced by the allochthonous subsidy provided by salmon and discuss implications for wolf-ungulate relations.