GC Concentration Research Articles

Biological Traits and Comprehensive Genomic Analysis of Novel Enterococcus faecalis Bacteriophage EFP6.

Enterococcus faecalis is a prevalent opportunistic pathogen associated with chicken embryonic and neonatal chick mortality, posing a significant challenge in poultry farming. In the current study, E. faecalis strain EF6, isolated from a recent hatchery outbreak, served as the host bacterium for the isolation of a novel phage EFP6, capable of lysing E. faecalis. Transmission electron microscopy revealed a hexagonal head and a short tail, classifying EFP6 as a member of the Autographiviridae family. EFP6 showed sensitivity to ultraviolet radiation and resistance to chloroform. The lytic cycle duration of EFP6 was determined to be 50 min, highlighting its efficacy in host eradication. With an optimal multiplicity of infection of 0.001, EFP6 exhibited a narrow lysis spectrum and strong specificity towards host strains. Additionally, EFP6 demonstrated optimal growth conditions at 40 °C and pH 8.0. Whole genome sequencing unveiled a genome length of 18,147 bp, characterized by a GC concentration of 33.21% and comprising 25 open reading frames. Comparative genomic assessment underscored its collinearity with related phages, notably devoid of lysogenic genes, thus ensuring genetic stability. This in-depth characterization forms the basis for understanding the biological attributes of EFP6 and its potential utilization in phage therapy, offering promising prospects for mitigating E. faecalis-associated poultry infections.

Glucocorticoids in preterm human milk.

BackgroundGlucocorticoids (GCs), cortisol and cortisone, are essential regulators of many physiological responses, including immunity, stress and mammary gland function. GCs are present in human milk (HM), but whether maternal and infant factors are associated with HM GC concentration following preterm birth is unclear.Materials and methodsHM samples were collected on postnatal day 5 and 10 and at 4 months’ corrected age (4m CA) in a cohort of moderate- and late-preterm infants. GCs in HM were measured by liquid chromatography-tandem mass spectrometry. Relationships between GCs in HM and both maternal and infant characteristics were investigated using Spearman’s correlations and linear mixed models.Results170 mothers of 191 infants provided 354 HM samples. Cortisol concentrations in HM increased from postnatal day 5–4m CA (mean difference [MD] 0.6 ± 0.1 ng/ml, p < 0.001). Cortisone concentration did not change across lactation but was higher than cortisol throughout. Compared to no antenatal corticosteroid (ANS), a complete course of ANS was associated with lower GC concentrations in HM through to 4m CA (cortisol: MD –0.3 ± 0.1 ng/ml, p < 0.01; cortisone MD –1.8 ± 0.4 ng/ml, p < 0.001). At 4m CA, higher maternal perceived stress was negatively associated with GC concentrations in HM (cortisol adjusted beta-coefficient [aβ] –0.01 ± 0.01 ng/ml, p = 0.05; and cortisone aβ –0.1 ± 0.03 ng/ml, p = 0.01), whereas higher postpartum depression and maternal obesity were associated with lower cortisone concentrations (aβ –0.1 ± 0.04 ng/ml p < 0.05; MD [healthy versus obese] –0.1 ± 0.04 ng/ml p < 0.05, respectively). There was a weak positive correlation between GC concentrations in HM and gestational age at birth (r = 0.1, p < 0.05). Infant birth head circumference z-score was negatively associated with cortisol concentrations (aβ –0.01 ± 0.04 ng/ml, p < 0.05). At hospital discharge, fat-free mass showed a weak positive correlation with cortisol concentrations (r = 0.2, p = 0.03), while fat mass showed a weak negative correlation with cortisone concentrations (r = –0.25, p < 0.001).ConclusionThe mammary gland appears to protect the infant from cortisol through inactivation into cortisone. Maternal and infant characteristics were associated with concentration of GCs in HM, including ANS, stress and depression scores, obesity, gestational age and infant size. The effects of HM glucocorticoids on long-term health outcomes requires further research.

Biophysical models unravel associations between glucocorticoids and thermoregulatory costs across avian species

Abstract Glucocorticoids (GCs) are metabolic hormones that regulate physiological and behavioural responses to environmental change and mediate homeostasis maintenance in vertebrates. Despite the assumption that GCs covary with energy metabolism, we yet lack a mechanistic understanding of how environmental factors such as temperature modulate GC variation through their effect on organismal energy balance. In particular, the mechanisms linking temperature‐dependent metabolic rate and GCs at broad spatial scales and across species remain poorly investigated. Here we used biophysical models to calculate thermoregulatory costs (i.e. the amount of heat required to keep body temperature in homeostasis) of free‐living birds as a function of environmental conditions, body size, shape and insulating layer of feathers. We then investigated the link between ambient temperature, cost of thermoregulation and baseline plasma GC concentrations in a comparative study including GC data from 94 bird species from HormoneBase. We found a significant, positive association between thermoregulatory costs and baseline GC concentrations. Interestingly, models including thermoregulatory costs better explained GC variation when compared to those including ambient temperature as a predictor variable. This result suggests that body size and shape fundamentally modulate energy requirements for thermoregulation and thereby GC concentrations in the wild. By providing a mechanistic description of the link between ambient temperature and thermoregulatory costs, biophysical models provide a tool to predict the impact of environmental conditions on energy metabolism. Our work demonstrates that differences in thermoregulation modulate variation in GC concentrations across a broad climatic gradient. A free Plain Language Summary can be found within the Supporting Information of this article.

Photosynthetic response of young oaks to biochar amendment in field conditions over 3 years

ABSTRACT Amendment by biochar made by thermal degradation of biomass is expected to enhance carbon sequestration through stimulating carbon assimilation by plants. We clarified the effect of biochar amendment on the photosynthesis of trees in forest ecosystems. Biochar was applied to young oak trees (Quercus serrata) in temperate deciduous forest at rates of 0, 5, 10 and 20 Mg ha−1 in four plots (C0, C5, C10, and C20). The variation in photosynthetic parameters (the maximum photosynthetic rate: Pmax, maximum carboxylation rate: Vcmax and the potential rate of electron transport: Jmax) and leaf traits (the stomatal conductance: gc, leaf mass per area (LMA) and leaf nutrient concentrations) were examined every month during the growing seasons for 3 years. Pmax generally increased in C5 and C10 and did not increase in C20. Similarly, Vcmax and Jmax increased in C5 and C10 and correlated significantly positively with Pmax, suggesting that biochar amendment basically increased the photosynthetic rate through improvements in physiological activities but that there was a maximum useful dosage. We also found that gc, LMA and leaf nutrient (N, Mg, and S) showed significant positive correlations with Pmax, indicating that an increase in photosynthetic rates would be supported by these leaf traits. However, stimulation of photosynthesis became smaller year by year, indicating that the effects of biochar amendment faded gradually. We concluded that biochar amendment basically improved the photosynthesis of oak trees in the forest through the change of all gc, LMA and leaf nutrient concentrations but declined yearly.

Biphasic Glucocorticoid Rhythm in One-Month-Old Infants: Reflection of a Developing HPA-Axis?

ContextThe hypothalamus-pituitary-adrenal (HPA) axis displays a diurnal rhythm. However, little is known about its development in early life.ObjectiveTo describe HPA-axis activity and study possible influencing factors in 1-month-old infants.DesignObservational.SettingAmsterdam University Medical Center, location VU University Medical Center (VUMC), and Onze Lieve Vrouwe Gasthuis (OLVG), Amsterdam.ParticipantsFifty-five mother-infant pairs.InterventionsCollection of breast milk and infants’ saliva 1 month postpartum for analysis of glucocorticoids (GCs; ie, cortisol and cortisone) using liquid chromatography– tandem mass spectrometry.Main Outcome MeasureGC rhythm in infants’ saliva and associations with vulnerability for maternal psychological distress (increased Hospital Anxiety and Depression Scale [HADS] score) or consultation at the Psychiatric Obstetric Pediatric (POP clinic), season at sampling, sex, and breast milk GC rhythmicity analyzed with SigmaPlot 14.0 software (Systat Software, San Jose, CA, USA) and regression analyses.ResultsA significant biphasic GC rhythm was detected in infants, with mean peaks [standard error of the mean, SEM] at 6:53 am [1:01] and 18:36 pm [1:49] for cortisol, and at 8:50 am [1:11] and 19:57 pm [1:13] for cortisone. HADS score, POP consultation, season at sampling, and sex were not associated with the infants’ GC rhythm. Breast milk cortisol maximum was positively associated with infants’ cortisol area-under-the-curve (AUC) increase and maximum. Higher breast milk cortisone AUC increase, AUC ground, and maximum were associated with an earlier maximum in infants. Breast milk and infant GC concentrations were associated between 6:00 am and 9:00 am.ConclusionsA biphasic GC rhythm, peaking in the morning and evening, was seen in 1-month-old infants at a group level. Breast milk GC parameters might be associated with the infants’ GC rhythm, possibly caused by a signaling effect of breast milk GCs, or as an associative effect of increased mother-infant synchrony. These results contribute to an increased understanding of early life HPA-axis development.

Fathers and sons: Physiological stress in male Zaisan mole voles, Ellobius tancrei

The social environment can be stressful for at least some group members, resulting in elevated levels of glucocorticoid stress hormones (GC). Patterns of the relationships between social rank and GC levels vary between species. In carnivores, primates and birds that live in permanent cooperative groups, helpers do not usually display physiological indicators of stress. Very little is known about status-related GC differences within cooperative groups of rodents. In this laboratory study, we compared GC concentrations in dominant (fathers) and subordinate (natal sons) males of a cooperative subterranean vole, Ellobius tancrei. The assessment of adrenocortical activity by measuring urine glucocorticoid metabolites (UGM) was previously validated for this species through an ACTH challenge test. We observed clear peaks of UGM in the second or third urine samples taken after the administration of ACTH (lag time equal to 2.5–3 h). Thus, UGM is suitable to estimate physiological stress in Ellobius. Postpubertal sons living in natal groups had significantly higher UGM concentrations than their fathers. The average UGM levels of sons were positively associated with their ages and paternal body masses, and negatively associated with paternal ages. Hence, son-father interactions rather than just younger ages of sons appear to contribute to GC differences. The revealed pattern was not consistent with that reported for most cooperative species from other taxa, highlighting the importance of comparative studies.

11β-HSD1 Modulates the Set Point of Brown Adipose Tissue Response to Glucocorticoids in Male Mice

Glucocorticoids (GCs) are potent regulators of energy metabolism. Chronic GC exposure suppresses brown adipose tissue (BAT) thermogenic capacity in mice, with evidence for a similar effect in humans. Intracellular GC levels are regulated by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) activity, which can amplify circulating GC concentrations. Therefore, 11β-HSD1 could modulate the impact of GCs on BAT function. This study investigated how 11β-HSD1 regulates the molecular architecture of BAT in the context of GC excess and aging. Circulating GC excess was induced in 11β-HSD1 knockout (KO) and wild-type mice by supplementing drinking water with 100 μg/mL corticosterone, and the effects on molecular markers of BAT function and mitochondrial activity were assessed. Brown adipocyte primary cultures were used to examine cell autonomous consequences of 11β-HSD1 deficiency. Molecular markers of BAT function were also examined in aged 11β-HSD1 KO mice to model lifetime GC exposure. BAT 11β-HSD1 expression and activity were elevated in response to GC excess and with aging. 11β-HSD1 KO BAT resisted the suppression of uncoupling protein 1 (UCP1) and mitochondrial respiratory chain subunit proteins normally imposed by GC excess. Furthermore, brown adipocytes from 11β-HSD1 KO mice had elevated basal mitochondrial function and were able to resist GC-mediated repression of activity. BAT from aged 11β-HSD1 KO mice showed elevated UCP1 protein and mitochondrial content, and a favorable profile of BAT function. These data reveal a novel mechanism in which increased 11β-HSD1 expression, in the context of GC excess and aging, impairs the molecular and metabolic function of BAT.

Elevated Hypothalamic Glucocorticoid Levels Are Associated With Obesity and Hyperphagia in Male Mice.

Glucocorticoid (Gc) excess, from endogenous overproduction in disorders of the hypothalamic-pituitary-adrenal axis or exogenous medical therapy, is recognized to cause adverse metabolic side effects. The Gc receptor (GR) is widely expressed throughout the body, including brain regions such as the hypothalamus. However, the extent to which chronic Gcs affect Gc concentrations in the hypothalamus and impact on GR and target genes is unknown. To investigate this, we used a murine model of corticosterone (Cort)-induced obesity and analyzed Cort levels in the hypothalamus and expression of genes relevant to Gc action. Mice were administered Cort (75 μg/mL) or ethanol (1%, vehicle) in drinking water for 4 weeks. Cort-treated mice had increased body weight, food intake, and adiposity. As expected, Cort increased plasma Cort levels at both zeitgeber time 1 and zeitgeber time 13, ablating the diurnal rhythm. Liquid chromatography dual tandem mass spectrometry revealed a 4-fold increase in hypothalamic Cort, which correlated with circulating levels and concentrations of Cort in other brain regions. This occurred despite decreased 11β-hydroxysteroid dehydrogenase (Hsd11b1) expression, the gene encoding the enzyme that regenerates active Gcs, whereas efflux transporter Abcb1 mRNA was unaltered. In addition, although Cort decreased hypothalamic GR (Nr3c1) expression 2-fold, the Gc-induced leucine zipper (Tsc22d3) mRNA increased, which indicated elevated GR activation. In keeping with the development of hyperphagia and obesity, Cort increased Agrp, but there were no changes in Pomc, Npy, or Cart mRNA in the hypothalamus. In summary, chronic Cort treatment causes chronic increases in hypothalamic Cort levels and a persistent elevation in Agrp, a mediator in the development of metabolic disturbances.

Training Reduces Stress in Human-Socialised Wolves to the Same Degree as in Dogs.

The welfare of animals in captivity is of considerable societal concern. A major source of stress, especially for wild animals, is the lack of control over their environment, which includes not being able to avoid contact with human beings. Paradoxically, some studies have shown that interactions with human beings may improve the welfare of wild animals in captivity. Here, we investigated the behavioural (behaviours indicative of cooperation or stress) and physiological (variations in salivary cortisol concentrations) effects of the increasingly used practice of training wild animals as a way to facilitate handling and/or as behavioural enrichment. We evaluated the effects of indoor training sessions with familiar caretakers on nine human-socialised individuals of a wild species, the wolf (Canis lupus), in comparison to nine individuals of its domesticated form, the dog (Canis lupus familiaris). All animals were raised and kept in intraspecific packs under identical conditions—in accordance with the social structure of the species—in order to control for socialisation with human beings and familiarity with training. We also collected saliva samples of trainers to measure GC and testosterone concentrations, to control for the effects of trainers’ stress levels on the responses of the animals. During the training sessions, separated from pack members, the animals stayed voluntarily close to the trainers and mostly adequately performed requested behaviours, indicating concentration to the task. Similarly to dogs, the salivary cortisol level of wolves–used as an index of stress—dropped during these sessions, pointing to a similar stress-reducing effect of the training interaction in both subspecies. The responses to the requested behaviours and the reduction in salivary cortisol level of wolves and dogs varied across trainers, which indicates that the relaxing effect of training has a social component. This points to another factor affecting the welfare of animals during the sessions, beside the rewarding effect of getting food and control over the situation by successfully completing a task. As all responses performed by the animals corresponded to cues already familiar to them, the reported effects were likely due to the above cited factors rather than to a learning process. Our results support previous findings suggesting that training is a potentially powerful tool for improving welfare in some wild social canids by creating structured and positive interactions between these animals and their human caretakers.

Characterizing predictors of survival in the American pika (Ochotona princeps)

The measurement of stress hormone (glucocorticoid [GC]) concentration is increasingly used to assess the health of wildlife populations. However, for many species, we do not have a good understanding of the range of GC concentrations that might indicate a compromised individual. A temporary increase in GC concentration can prompt the adoption of behavior or activities to promote individual survival. However, chronic GC elevation results in deleterious effects on health that can reduce survival. In order to use GC concentration as a metric of individual fitness for a given species, it will be necessary to relate individual demographic rates to GC concentration. We related survival in American pikas ( Ochotona princeps ) to 2 different stress metrics, glucocorticoid metabolite (GCM) concentration in fecal samples and GC concentration in plasma samples. Annual survival was analyzed in relation to each of these stress metrics as well as other physiological metrics and habitat characteristics at several sites in the Rocky Mountains. Among the predictors considered, GCM concentration was by far the strongest predictor of annual survival in pikas, and individuals with higher baseline GCM were less likely to survive. Our metric of flea load was also negatively related to annual survival. Given the limited time and resources that characterize many wildlife conservation projects, it is important to establish which endocrine metrics are the most informative for a species. American pikas have been identified as a sentinel species for detecting effects of climate change, and several correlational studies have projected range contraction for the species. Our results suggest that more mechanistic projections might be possible given further study of the relationship between GCM and climate. Our approach contributes to a better understanding of factors affecting survival in this species and provides a basis for further research relating individual stress response and survival to environmental change.

Maternal Behavior and Physiological Stress Levels in Wild Chimpanzees (Pan troglodytes schweinfurthii).

Individual differences in maternal behavior toward, and investment in, offspring can have lasting consequences, particularly among primate taxa characterized by prolonged periods of development over which mothers can exert substantial influence. Given the role of the neuroendocrine system in the expression of behavior, researchers are increasingly interested in understanding the hormonal correlates of maternal behavior. Here, we examined the relationship between maternal behavior and physiological stress levels, as quantified by fecal glucocorticoid metabolite (FGM) concentrations, in lactating chimpanzees, Pan troglodytes schweinfurthii, at Gombe National Park, Tanzania. After accounting for temporal variation in FGM concentrations, we found that mothers interacted socially (groomed and played) with and nursed their infants more on days when FGM concentrations were elevated compared to days when FGM concentrations were within the range expected given the time of year. However, the proportion of time mothers and infants spent in contact did not differ based on FGM concentrations. These results generally agree with the suggestion that elevated GC concentrations are related to maternal motivation and responsivity to infant cues and are the first evidence of a hormonal correlate of maternal behavior in a wild great ape.

Albuminoid genes: evolving at the interface of dispensability and selection.

The albuminoid gene family comprises vitamin D-binding protein (GC), alpha-fetoprotein (AFP), afamin (AFM), and albumin (ALB). Albumin is the most abundant human serum protein, and, as the other family members, acts as a transporter of endogenous and exogenous substances including thyroxine, fatty acids, and drugs. Instead, the major cargo of GC is 25-hydroxyvitamin D. We performed an evolutionary study of albuminoid genes and we show that ALB evolved adaptively in mammals. Most positively selected sites are located within albumin-binding sites for fatty acids and thyroxine, as well as at the contact surface with neonatal Fc receptor. Positive selection was also detected for residues forming the prostaglandin-binding pocket. Adaptation to hibernation/torpor might explain the signatures of episodic positive selection we detected for few mammalian lineages. Application of a population genetics–phylogenetics approach showed that purifying selection represented a major force acting on albuminoid genes in both humans and chimpanzees, with the strongest constraint observed for human GC. Population genetic analysis revealed that GC was also the target of locally exerted selective pressure, which drove the frequency increase of different haplotypes in distinct human populations. A search for known variants that modulate GC and 25-hydroxyvitamin D concentrations revealed linkage disequilibrium with positively selected variants, although European and Asian major GC haplotypes carry alleles with reported opposite effect on GC concentration. Data herein indicate that albumin, an extremely abundant housekeeping protein, was the target of pervasive and episodic selection in mammals, whereas GC represented a selection target during the recent evolution of human populations.

White Shrimp Litopenaeus vannamei that Have Received Carrageenan via Immersion Show Protective Immunity after Salinity Stress

White shrimp ＂Litopenaeus vannamei＂ which had been immersed in seawater (35 per mille) containing carrageenan at 0 (control) and 200 mg/l for 3 h, were subjected to salinity change (transferred to 25 per mille), and the immune parameters including hyaline cells (HCs), granular cells (GCs, including semi-granular cells), total hemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, hemolymph protein concentration were examined 6~96 h post-transfer. Shrimp with no exposure to carrageenan and no salinity change served as the background control. Results indicated that HC, GC, THC, PO activity, RB, SOD activity, and hemolymph protein concentration of shrimp immersed in 200 mg/l carrageenan were significantly higher than those of control shrimp at 6~12 h post-stress. Results also indicated that these parameters of shrimp immersed in 200 mg/l carrageenan had returned to the background values at 24, 24, 24, 48, 12, 48, and 12 h post-stress, whereas these immune parameters in control shrimp had returned to the original values at 96 h post-stress. It was therefore concluded that the innate immunity of shrimp which had been immersed in seawater containing the carrageenan exhibited a protective effect against low salinity change as evidenced by earlier recovery of immune parameters.

The Cytosolic Nucleoprotein of the Plant-Infecting Bunyavirus Tomato Spotted Wilt Recruits Endoplasmic Reticulum–Resident Proteins to Endoplasmic Reticulum Export Sites

In contrast with animal-infecting viruses, few known plant viruses contain a lipid envelope, and the processes leading to their membrane envelopment remain largely unknown. Plant viruses with lipid envelopes include viruses of the Bunyaviridae, which obtain their envelope from the Golgi complex. The envelopment process is predominantly dictated by two viral glycoproteins (Gn and Gc) and the viral nucleoprotein (N). During maturation of the plant-infecting bunyavirus Tomato spotted wilt, Gc localizes at endoplasmic reticulum (ER) membranes and becomes ER export competent only upon coexpression with Gn. In the presence of cytosolic N, Gc remains arrested in the ER but changes its distribution from reticular into punctate spots. Here, we show that these areas correspond to ER export sites (ERESs), distinct ER domains where glycoprotein cargo concentrates prior to coat protein II vesicle-mediated transport to the Golgi. Gc concentration at ERES is mediated by an interaction between its cytoplasmic tail (CT) and N. Interestingly, an ER-resident calnexin provided with Gc-CT was similarly recruited to ERES when coexpressed with N. Furthermore, disruption of actin filaments caused the appearance of a larger amount of smaller ERES loaded with N-Gc complexes, suggesting that glycoprotein cargo concentration acts as a trigger for de novo synthesis of ERES.

Triplet entropy analysis of hemagglutinin and neuraminidase sequences measures influenza virus phylodynamics

The influenza virus has been a challenge to science due to its ability to withstand new environmental conditions. Taking into account the development of virus sequence databases, computational approaches can be helpful to understand virus behavior over time. Furthermore, they can suggest new directions to deal with influenza. This work presents triplet entropy analysis as a potential phylodynamic tool to quantify nucleotide organization of viral sequences. The application of this measure to segments of hemagglutinin (HA) and neuraminidase (NA) of H1N1 and H3N2 virus subtypes has shown some variability effects along timeline, inferring about virus evolution. Sequences were divided by year and compared for virus subtype (H1N1 and H3N2). The nonparametric Mann–Whitney test was used for comparison between groups. Results show that differentiation in entropy precedes differentiation in GC content for both groups. Considering the HA fragment, both triplet entropy as well as GC concentration show intersection in 2009, year of the recent pandemic. Some conclusions about possible flu evolutionary lines were drawn.

New insight on the molecular aspects of glucocorticoid effects in nervous system development.

Adrenal glucocorticoids (Gc) are among the most significant hormones in the mammalian organisms; these steroids may reach and penetrate all tissues where they interact with cytoplasmic/nuclear receptors, through which they exert multiple and very multifaceted actions. The effects of physiological concentrations of Gc on brain functions have not been completely clarified, even though Gc are recognized to influence behavioral responses, emotions, cognitive processes and to take part in the neuroendocrine control of body homeostasis. Developmental programming effects of Gc in animal models and humans have been proposed. Actually, pre-natal stress, or exposure to high Gc levels, would somehow affect neuronal developmental events in some structure and this can lead to central nervous system altered functions, as the impairment of neuroendocrine activities, cognitive processes, sleep and mood disorders. Interestingly, it has been observed that these abnormalities may not be limited to the first directly exposed individuals but transmissible across generations. The establishment of animal models with localized pre-natal glucocorticoid receptors deficiency led to the accumulation of data on the possible roles of these hormones on development of the central and peripheral nervous system. The most recent findings on the effects of Gc on neuroblast development, with particular attention to neuronal migration, will be presented.

Mediating free glucocorticoid levels in the blood of vertebrates: are corticosteroid‐binding proteins always necessary?

Summary Glucocorticoids (GC) are integral to the stress response of vertebrates to environmental challenges. Their impact is immediate and widespread, and prolonged exposure can result in major activational and organizational changes. The vertebrate body has two mechanisms to limit GC impact: first, a rapid negative feedback system to turn off their release, and second, a protein – corticosteroid‐binding globulin (CBG) – to prevent them being free in the blood. Species used in biomedical research have CBG levels that normally bind 90–95% of blood GC. This evidence was the basis for the ‘Free Hormone Hypothesis,’ which posits that only unbound, free hormone is available for use by tissues and is biologically active. High levels of free GC typically occur only under conditions of chronic stress and are associated with major suppression of key body functions. The hypothesis proposes that the primary role of CBG is to render GCs unavailable, thereby preventing tissue exposure. From a field study in southern Ontario on northern (Glaucomys sabrinus) and southern (Glaucomys volans) flying squirrels, we found virtually no CBG binding capacity in their plasma, resulting in only about 10% of cortisol being bound throughout the year. However, neither species showed any evidence of being physiologically compromised. This presents a major challenge to the potential consequences of chronically high levels of free GC. To assess the generality of this finding, we carried out a phylogenetic comparison of 91 vertebrate species in which both GC and CBG levels were measured. In 93%, CBG levels were sufficient to bind about 90% of circulating GCs. This evidence is thus concordant with that from biomedical research. However, both flying squirrel species and four species of New World monkeys were extreme outliers. These two groups had the highest GC concentrations, but relatively little binding capacity (10% or less of their GC was bound). An ancestor state reconstruction of the proportion of GCs bound indicated that the flying squirrels and New World monkeys evolved this character state from ancestors that followed the common 90% bound pattern. It also indicated that the original vertebrates – the earliest fishes – have most of their GC in the free state and little or no steroid‐binding protein. We thus demonstrate a dichotomous pattern with respect to CBG: a dominant branch, where high levels of CBG bind most of the GC, applies to the majority of vertebrates; and a secondary branch, where low levels of CBG bind almost none of the GC, applies to a very small subset. For the latter, the critical unknown is how these species mitigate the impact of the high free GC levels and how such a dramatic trait shift could evolve.

Increasing the Isolated Quantities and Purities of Volatile Compounds by Using a Triple Deans-Switch Multidimensional Preparative Gas Chromatographic System with an Apolar-Wax-Ionic Liquid Stationary-Phase Combination

A novel preparative-scale triple gas chromatographic system, equipped with three Deans-switch devices, is herein described. The isolation of volatile compounds was achieved by using a lab-made collection system. Considerations are made on the possibility to inject high volumes of neat sample, maintaining, at the same time, high resolution levels. The degree of recovery of a series of compounds, characterized by a differing volatility, is also reported. In this respect, different collection conditions were evaluated, namely, the presence (or not) of packing material as well as CO(2) cooling, in various combinations. Finally, the interconnected features, that is time requirements and the isolation of "acceptable" analyte quantities (e.g., milligram level for NMR characterization), are related to parameters such as analyte concentration, injected volume, collection conditions, and number of GC runs. The results herein reported will demonstrate the need for a high-resolution GC step, prior to analyte collection, in the prep-GC analysis of complex samples.

Can mailed swab samples be dry-shipped for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis by nucleic acid amplification tests?

Dry-shipped and mailed vaginal swabs collected at home have been used in research studies for the detection of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (GC), and Trichomonas vaginalis (TV) by nucleic acid amplification tests (NAATs) in screening programs. A verification study was performed to compare the limit of detection of CT, GC, and TV on swabs that were dry-shipped to paired swabs that were wet-shipped in transport media through the US mail. The Centers for Disease Control and Prevention prepared inocula in sterile water to mock simulated urogenital swabs with high to low concentrations of CT and GC. Replicate swabs were inoculated with 100 μL of dilutions and were dry transported or placed into commercial transport media (“wet”) for mailing for NAAT testing. The University of Alabama prepared replicate concentrations of TV, which were similarly shipped and tested by NAAT. All paired dry and wet swabs were detectable for CT. For GC, all paired dry and wet swabs were detectable for GC at concentrations ≥103. At 102 and 10 CFU/mL, the 10 replicate GC results were variably positive. For TV, wet and dry shipped concentrations >102 TV/mL tested positive, while results at 10 TV/mL were negative for dry swabs. Holding replicate dry swabs at 55 ○C 5 days before testing did not affect results. NAATs were able to detect CT, GC, and TV on dry transported swabs. Using NAATs for testing home-collected, urogenital swabs mailed in a dry state to a laboratory may be useful for outreach screening programs.

Relationship between Group-Specific Component Protein and the Development of Asthma

Airway inflammation and remodeling during asthma are attributed to the altered expression of biologically relevant proteins. To search for asthma-specific proteins in bronchoalveolar lavage fluid (BAL) from individuals with asthma and to validate the identified proteins in an experimental model of asthma. Liquid chromatography-tandem mass spectrometry was performed to identify proteins in BAL fluid found by two dimensional electrophoresis (2DE) to be differentially expressed in subjects with asthma versus control subjects. Group-specific component (Gc) and mRNA levels were measured using an ELISA, Western blots, and PCR. A neutralization study using an antibody against Gc protein was performed in an experimental asthma model. Based on 2DE, 15 proteins were significantly up-regulated or down-regulated in eight subjects with asthma compared with eight control subjects. The protein levels of Gc, hemopexin, and haptoglobin-b were increased, whereas the a1- antitrypsin and glutathione S-transferase levels were decreased in subjects with asthma. The Gc concentration in BAL fluid was significantly elevated in 67 subjects with asthma compared with that in 22 control subjects (P < 0.009). The Gc was significantly correlated with the neutrophil percentage in BAL fluid of subjects with asthma (P = 0.001). Gc mRNA and protein levels were higher in ovalbumin-sensitized/ challenged asthma mice than in sham-treated mice. Gc protein were expressed on alveolar macrophages and on epithelial cells. Treatment with an anti-Gc antibody dose-dependently reduced the ovalbumin sensitization/challenge-induced enhancement of airway hyperreactivity, airway inflammation, goblet cell hyperplasia,and levels of eotaxin, interleukin-4, -5, and -13, and interferon-g. Gc may be involved in the development of asthma, and the neutralization of Gc protein could be a therapeutic strategy for asthma.