A protocol for distilling animal body water from biological samples and measuring oxygen and hydrogen stable isotopes via cavity ring-down spectroscopy.

The application of stable isotope analysis (SIA) to the fields of ecology and animal biology has rapidly expanded over the past three decades, particularly with regards to water analysis. SIA now provides the opportunity to monitor migration patterns, examine food webs, and assess habitat changes in current and past study systems. While carbon and nitrogen SIA of biological samples have become common, analyses of oxygen or hydrogen are used more sparingly despite their promising utility for tracing water sources and animal metabolism. Common ecological applications of oxygen or hydrogen SIA require injecting enriched isotope tracers. As such, methods for processing and analyzing biological samples are tailored for enriched tracer techniques, which require lower precision than other techniques given the large signal-to-noise ratio of the data. However, instrumentation advancements are creating new opportunities to expand the applications of high-throughput oxygen and hydrogen SIA. To support these applications, we update methods to distill and measure water derived from biological samples with consistent precision equal to, or better than, ± 0.1 ‰ for δ17O, ± 0.3 ‰ for δ18O, ± 1 ‰ for δ2H, ± 2 ‰ for d-excess, and ± 15 per meg for Δ17O.

Water restriction increases oxidation of endogenous amino acids in house sparrows (Passer domesticus).

Animals can cope with dehydration in a myriad of ways, both behaviorally and physiologically. The oxidation of protein produces more metabolic water per kilojoule than that of fat or carbohydrate, and it is well established that birds increase protein catabolism in response to high rates of water loss. However, the fate of amino acids mobilized in response to water restriction has not been explicitly determined. While protein catabolism releases bound water, we hypothesized that water-restricted birds would also oxidize the resulting amino acids, producing additional water as a product of oxidative phosphorylation. To test this, we fed captive house sparrows (Passer domesticus) 13C-labeled leucine for 9 weeks to label endogenous proteins. We conducted weekly trials during which we measured the physiological response to water restriction as changes in lean mass, fat mass, metabolism and the enrichment of 13C in exhaled CO2 (δ13Cbreath). If water-restricted birds catabolized proteins and oxidized the resulting amino acids, we expected to simultaneously observe greater lean mass loss and elevated δ13Cbreath relative to control birds. We found that water-restricted birds catabolized more lean tissue and also had enriched δ13Cbreath in response to water restriction, supporting our hypothesis. δ13Cbreath, however, varied with metabolic rate and the length of the water restriction period, suggesting that birds may spare protein when water balance can be achieved using other physiological strategies.

Relating wing morphology and immune function to patterns of partial and differential bat migration using stable isotopes.

Migration is energetically expensive and is predicted to drive similar morphological adaptations and physiological trade-offs in migratory bats and birds. Previous studies suggest that fixed traits like wing morphology vary among species and individuals according to selective pressures on flight, while immune defences can vary flexibly within individuals as energy is variably reallocated throughout the year. We assessed intraspecific variation in wing morphology and immune function in silver-haired bats Lasionycteris noctivagans, a species that follows both partial and differential migration patterns. We hypothesized that if bats experience energy constraints associated with migration, then wing morphology and immune function should vary based on migratory tendency (sedentary or migratory) and migration distance. We predicted that long-distance migrants would have reduced immune function and more migration-adapted wing shapes compared to resident or short-distance migrating bats. We estimated breeding latitude of spring migrants using stable hydrogen isotope techniques. Our sample consisted primarily of male bats, which we categorized as residents, long-distance northern migrants, short-distance northern migrants and southern migrants (apparent breeding location south of capture site). Controlling for individual condition and capture date, we related wing characteristics and immune indices among groups. Some, but not all, aspects of wing form and immune function varied between migrants and residents. Long-distance northern migrants had larger wings than short-distance northern migrants and lower wing loading than southern migrants. Compared with resident bats, short-distance northern migrants had reduced IgG while southern migrants had heightened neutrophils and neutrophil-to-lymphocyte ratios. Body fat, aspect ratio, wing tip shape and bacteria killing ability did not vary with migration status or distance. In general, male silver-haired bats do not appear to mediate migration costs by substantially downregulating immune defences or to be under stronger selection for wing forms adapted for fast, energy-efficient flight. Such phenotypic changes may be more adaptive for female silver-haired bats, which migrate farther and are more constrained by time in spring than males. Adaptations for aerial hawking and the use of heterothermy by migrating bats may also reduce the energetic cost of migration and the need for more substantial morphological and physiological trade-offs.

Migration and reproduction are associated with similar degrees of phenotypic flexibility in an insectivorous bat.

As organisms face variation in energetic challenges and physiological demands, they often respond with reversible changes in behavior, physiology, and morphology, described as phenotypic flexibility. From the magnitude of phenotypic change, we can infer the energetic challenges of different life stages. We studied phenotypic flexibility in a population of reproductive and pre-migratory female insectivorous bats (Tadarida brasiliensis). While female reproductive demands are well described in insectivorous bats, there are questions regarding the demands of migration. Our objective was to measure phenotypic flexibility to assess the cost of autumn migration compared to reproduction in an insectivorous bat. We measured plasma triglycerides to quantify foraging rate, and body composition (body mass and individual organ mass) of T. brasiliensis throughout the summer season (from arrival in spring through pre-migration/migration departure in autumn) according to the female reproductive cycle. We found phenotypic changes during pre-migration/migration similar to periods of high-energy demand during reproduction (e.g., late pregnancy and lactation). Most notably, bats weighed as much during peak pregnancy, as they did during migration, and the rapid mass gain from post-lactation through the migratory period was due to a combination of hyperphagia and hypertrophy of digestive organs. Our results indicate that energetic demands incurred during migration are similar to those during reproduction and emphasize the energetic challenges of migration.

Seasonal Dynamics of Lipid Metabolism and Energy Storage in the Brazilian Free-Tailed Bat.

As small, flying, mammalian endotherms, insectivorous bats are adapted to operate at high levels of energy expenditure. In response to seasonally variable challenges, we predicted that bats should balance energy budgets by flexibly adjusting aspects of their physiology or behavior in ways that elevate metabolic capacity. We examined variation in energy storage and pathways for oxidative metabolism in Brazilian free-tailed bats (Tadarida brasiliensis) related to estimated costs associated with reproduction and migration. We collected pectoral muscle and liver from female T. brasiliensis at six time points during the summer and fall and measured changes in the activity of four enzymes involved with lipid metabolism. Body mass varied substantially with life-cycle stage, suggesting that rapid accumulation and use of fat stores occurs in response to current and anticipated energy demands. Catabolic enzyme activity (carnitine palmitoyl transferase [CPT], 3-hydroxyacyl-CoA dehydrogenase [HOAD], and citrate synthase [CS]) in the muscle was increased during lactation compared with early pregnancy but exhibited no change before fall migration. While there was no temporal change in lipid biosynthetic capacity in the liver, fatty acid synthase activity was negatively correlated with body mass. Variation in body mass and enzyme activity in T. brasiliensis during the summer suggests that stored energy is mobilized and lipid oxidative capacity is increased during periods of increased demand and that lipid biosynthetic capacity is increased with depletion of fat stores. These results suggest that bats are able to flexibly adjust metabolic capacity based on energy requirement to maintain energy balance despite high levels of expenditure.

Reliability and validity of the Brief 2-Way Social Support Scale: an investigation of social support in promoting older adult well-being.

OBJECTIVES: This study aimed to explore the relationship between well-being and perceived stress, and the functional dimensions of social support in older adults. METHOD: Data from 306 older adults were obtained in a survey containing the two-way Social Support Scale (2-Way SSS). Also, a subset of the sample (N = 165) was filled out with measures of well-being and perceived stress, and a follow-up survey was completed 3 months later (N = 111). RESULTS: Confirmatory factor analyses and reliability analyses provide evidence for a 12-item Brief 2-Way SSS as a reliable and valid measure of the four domains of Social Support. Correlations and regression analyses indicated the scale displayed good concurrent and predictive validity across time points, with receiving emotional support positively associated with well-being at Time 1 (T1) and Time 2 (T2), and Receiving Instrumental Support negatively associated with perceived stress at TI and T2. CONCLUSIONS: This study provides support for the importance of examining the influence of separable elements of social support on psychological outcomes in older adults. The Brief 2-Way SSS was found to have good psychometric properties in this sample of older adults.

The White-Nose Syndrome Transcriptome: Activation of Anti-fungal Host Responses in Wing Tissue of Hibernating Little Brown Myotis.

White-nose syndrome (WNS) in North American bats is caused by an invasive cutaneous infection by the psychrophilic fungus Pseudogymnoascus destructans (Pd). We compared transcriptome-wide changes in gene expression using RNA-Seq on wing skin tissue from hibernating little brown myotis (Myotis lucifugus) with WNS to bats without Pd exposure. We found that WNS caused significant changes in gene expression in hibernating bats including pathways involved in inflammation, wound healing, and metabolism. Local acute inflammatory responses were initiated by fungal invasion. Gene expression was increased for inflammatory cytokines, including interleukins (IL) IL-1ß, IL-6, IL-17C, IL-20, IL-23A, IL-24, and G-CSF and chemokines, such as Ccl2 and Ccl20. This pattern of gene expression changes demonstrates that WNS is accompanied by an innate anti-fungal host response similar to that caused by cutaneous Candida albicans infections. However, despite the apparent production of appropriate chemokines, immune cells such as neutrophils and T cells do not appear to be recruited. We observed upregulation of acute inflammatory genes, including prostaglandin G/H synthase 2 (cyclooxygenase-2), that generate eicosanoids and other nociception mediators. We also observed differences in Pd gene expression that suggest host-pathogen interactions that might determine WNS progression. We identified several classes of potential virulence factors that are expressed in Pd during WNS, including secreted proteases that may mediate tissue invasion. These results demonstrate that hibernation does not prevent a local inflammatory response to Pd infection but that recruitment of leukocytes to the site of infection does not occur. The putative virulence factors may provide novel targets for treatment or prevention of WNS. These observations support a dual role for inflammation during WNS; inflammatory responses provide protection but excessive inflammation may contribute to mortality, either by affecting torpor behavior or causing damage upon emergence in the spring.

Allosteric regulation of Argonaute proteins by miRNAs.

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) bind to Argonaute (AGO) family proteins to form a related set of effector complexes that have diverse roles in post-transcriptional gene regulation throughout the eukaryotic lineage. Here sequence and structural analysis of the MID domain of the AGO proteins identified similarities with a family of allosterically regulated bacterial ligand-binding domains. We used in vitro and in vivo approaches to show that certain AGO proteins (those involved in translational repression) have conserved this functional allostery between two distinct sites, one involved in binding miRNA-target duplex and the other in binding the 5' cap feature (m(7)GpppG) of eukaryotic mRNAs. This allostery provides an explanation for how miRNA-bound effector complexes may avoid indiscriminate repressive action (mediated through binding interactions with the cap) before full target recognition.

Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome.

The fidelity of aminoacyl-tRNA selection by the ribosome depends on a conformational switch in the decoding center of the small ribosomal subunit induced by cognate but not by near-cognate aminoacyl-tRNA. The aminoglycosides paromomycin and streptomycin bind to the decoding center and induce related structural rearrangements that explain their observed effects on miscoding. Structural and biochemical studies have identified ribosomal protein S12 (as well as specific nucleotides in 16S ribosomal RNA) as a critical molecular contributor in distinguishing between cognate and near-cognate tRNA species as well as in promoting more global rearrangements in the small subunit, referred to as "closure." Here we use a mutational approach to define contributions made by two highly conserved loops in S12 to the process of tRNA selection. Most S12 variant ribosomes tested display increased levels of fidelity (a "restrictive" phenotype). Interestingly, several variants, K42A and R53A, were substantially resistant to the miscoding effects of paromomycin. Further characterization of the compromised paromomycin response identified a probable second, fidelity-modulating binding site for paromomycin in the 16S ribosomal RNA that facilitates closure of the small subunit and compensates for defects associated with the S12 mutations.

A variation of the translation attenuation model can explain the inducible regulation of the pBC16 tetracycline resistance gene in Bacillus subtilis.

Expression of the tet resistance gene from plasmid pBC16 is induced by the antibiotic tetracycline, and induction is independent of the native promoter for the gene. The nucleotide sequence at the 5' end of the tet mRNA (the leader region) is predicted to assume a complex secondary structure that sequesters the ribosome binding site for the tet gene. A spontaneous, constitutively expressed tet gene variant contains a mutation predicted to provide the tet gene with a nonsequestered ribosome binding site. Lastly, comparable levels of tet mRNA can be demonstrated in tetracycline-induced and uninduced cells. These results are consistent with the idea that the pBC16 tet gene is regulated by translation attenuation, a model originally proposed to explain the inducible regulation of the cat and erm genes in gram-positive bacteria. As with inducible cat and erm genes, the pBC16 tet gene is preceded by a translated leader open reading frame consisting of a consensus ribosome binding site and an ATG initiation codon, followed by 19 sense codons and a stop codon. Mutations that block translation of cat and erm leaders prevent gene expression. In contrast, we show that mutations that block translation of the tet leader result in constitutive expression. We provide evidence that translation of the tet leader peptide coding region blocks tet expression by preventing the formation of a secondary-structure complex that would, in the absence of leader translation, expose the tet ribosome binding site. Tetracycline is proposed to induce tet by blocking or slowing leader translation. The results indicate that tet regulation is a variation of the translation attenuation model.

Barriers to adequate nutrition in critically ill children.

OBJECTIVE: The study assessed the adequacy of nutrition support in critically ill infants and children and identifies barriers impeding the delivery of estimated energy requirement (EER). METHODS: Forty-two children (median age, 6.6 mo; range, 0-198) who were admitted to a tertiary-level pediatric intensive care unit (PICU) were studied prospectively over a 6-mo period. Patients staying in the PICU longer than a full 3 d and who received enteral or a combination of enteral and parenteral nutrition were eligible for inclusion. Patients were assigned to one of two groups: patients after cardiac surgery (n = 18) and all other diagnoses (n = 24). EERs were compared with actual energy intake, and clinical information was collected throughout the PICU admission. RESULTS: Patients in the PICU received a median of 37.7% (range, 0.2-130.2%) of their EERs. The cardiac group achieved significantly lower energy intakes than did the non-cardiac group (P = 0.02). Only 22 of 42 patients (52%) achieved full EERs at any time during their admission, and this was more likely in non-cardiac patients (67% versus 33%, P = 0.03) Children undergoing cardiac surgery had a significant fall in weight-for-age Z scores (WAZ) from PICU admission to discharge (median WAZ, -1.44 versus -2.14; P < 0.001). In both groups, the major barrier to achieving EER was fluid volume restriction. Interruption of feeding for procedures and feeding intolerance reduced energy intake to a lesser degree. CONCLUSIONS: This study highlights the inadequacy of nutrition support in critically ill children in the PICU. Restriction of fluid intake was the main barrier to the delivery of adequate nutrition, particularly in infants undergoing cardiac surgery.

The chloramphenicol-inducible catB gene in Agrobacterium tumefaciens is regulated by translation attenuation.

Agrobacterium tumefaciens strains C58, A136, and BG53 are chloramphenicol resistant, and each contains the catB gene originally identified by Tennigkeit and Matzuran (Gene 99:113-116, 1991). The chloramphenicol acetyltransferase activity in all of the strains is chloramphenicol inducible. Examination of the catB gene in strain BG53 indicates that it is regulated by an attenuation mechanism similar to translation attenuation that regulates inducible catA genes resident in gram-positive bacteria and the inducible cmlA gene that confers chloramphenicol resistance in Pseudomonas spp.