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Urbanization drives phenotypic variation in many animal species. This includes behavioral and physiological traits such as activity patterns, aggression, and hormone levels. A current challenge of urban evolutionary ecology is to understand the environmental drivers of phenotypic variation in cities. Moreover, do individuals develop tolerance to urban environmental factors, which underlie adaptative responses and contribute to the evolution of urban populations? Most available evidence comes from correlative studies and rare experiments where a single urban-related environmental factor has been manipulated in the field. Here we present the results of an experiment in which we tested for differences in the glucocorticoid (CORT) response of urban and rural blue tits nestlings (Cyanistes caeruleus) to artificial light at night (ALAN). ALAN has been suggested to alter CORT response in several animal species, but to date no study has investigated whether this effect of ALAN differs between urban and rural populations. Immediately after hatching, urban and forest broods were either exposed to 2 lux of ALAN (using an LED source mounted inside the nestbox) or received no treatment (dark control). The experiment lasted until the chicks fledged. When the chicks were 13 days old plasma samples were collected to measure baseline CORT concentrations, and feather samples to provide an integrative measure of CORT during growth. Forest birds had higher plasma CORT (pCORT) concentrations than their urban counterparts, irrespective of whether they were exposed to ALAN or not. Conversely, we found population-specific responses of feather CORT to ALAN. Specifically, urban birds that received ALAN had increased feather CORT compared with the urban dark controls, while the opposite was true for the forest birds. pCORT concentrations were negatively associated to fledging success, irrespective of population and treatment, while feather CORT was positively associated to fledging success in broods exposed to ALAN, but negatively in the dark control ones. Our results demonstrate that ALAN can play a role in determination of the glucocorticoid phenotype of wild animals, and may thus contribute to phenotypic differences between urban and rural animals., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

The glucocorticoid receptor is a key regulator of essential physiological processes, which under the control of the Hsp90 chaperone machinery, binds to steroid hormones and steroid-like molecules and in a rather complicated and elusive response, regulates a set of glucocorticoid responsive genes. We here examine a human glucocorticoid receptor variant, harboring a point mutation in the last C-terminal residues, L773P, that was associated to Primary Generalized Glucocorticoid Resistance, a condition originating from decreased affinity to hormone, impairing one or multiple aspects of GR action. Using in vitro and in silico methods, we assign the conformational consequences of this mutation to particular GR elements and report on the altered receptor properties regarding its binding to dexamethasone, a NCOA-2 coactivator-derived peptide, DNA, and importantly, its interaction with the chaperone machinery of Hsp90.

Adhesion G-protein-coupled receptors (GPCRs) are a major family of GPCRs, but limited knowledge of their ligand regulation or structure is available 1-3 . Here we report that glucocorticoid stress hormones activate adhesion G-protein-coupled receptor G3 (ADGRG3; also known as GPR97) 4-6 , a prototypical adhesion GPCR. The cryo-electron microscopy structures of GPR97-G o complexes bound to the anti-inflammatory drug beclomethasone or the steroid hormone cortisol revealed that glucocorticoids bind to a pocket within the transmembrane domain. The steroidal core of glucocorticoids is packed against the 'toggle switch' residue W 6.53 , which senses the binding of a ligand and induces activation of the receptor. Active GPR97 uses a quaternary core and HLY motif to fasten the seven-transmembrane bundle and to mediate G protein coupling. The cytoplasmic side of GPR97 has an open cavity, where all three intracellular loops interact with the G o protein, contributing to the high basal activity of GRP97. Palmitoylation at the cytosolic tail of the G o protein was found to be essential for efficient engagement with GPR97 but is not observed in other solved GPCR complex structures. Our work provides a structural basis for ligand binding to the seven-transmembrane domain of an adhesion GPCR and subsequent G protein coupling.

The photodegradation process of seven glucocorticoids (GCs), cortisone (CORT), hydrocortisone (HCORT), betamethasone (BETA), dexamethasone (DEXA), prednisone (PRED), prednisolone (PREDLO) and triamcinolone (TRIAM) was studied in tap and river water at a concentration close to the environmental ones. All drugs underwent sunlight degradation according to a pseudo-first-order decay. The kinetic constants ranged from 0.00082 min -1 for CORT to 0.024 min -1 for PRED and PREDLO. The photo-generated products were identified by high-pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). The main steps of the degradation pathways were the oxidative cleavage of the chain 17 for CORT, HCORT and the rearrangement of the cyclohexadiene moiety for the other GCs. The acute and chronic toxicity of GCs and of their photoproducts was assessed by the V. fischeri and P. subcapitata inhibition assays. The bioassays revealed no significant differences in toxicity between the parent compounds and their photoproducts, but the two organisms showed different responses. All samples produced a moderate acute toxic effect on V. fisheri and no one in the chronic tests. On the contrary, evident hormesis or eutrophic effect was produced on the algae, especially for long-term contact.

Glucocorticoids are widely used anti-inflammatory drugs in clinical settings. However, they can induce skeletal muscle atrophy by reducing fiber cross-sectional area and myofibrillar protein content. Studies have proven that antioxidants can improve glucocorticoid-induced skeletal muscle atrophy. Quercetin is a potent antioxidant flavonoid widely distributed in fruits and vegetables and has shown protective effects against dexamethasone-induced skeletal muscle atrophy. In this study, we demonstrated that dexamethasone significantly inhibited cell growth and induced cell apoptosis by stimulating hydroxyl free radical production in C2C12 skeletal muscle cells. Our results evidenced that quercetin increased C2C12 skeletal cell viability and exerted antiapoptotic effects on dexamethasone-treated C2C12 cells by regulating mitochondrial membrane potential (ΔΨm) and reducing oxidative species. Quercetin can protect against dexamethasone-induced muscle atrophy by regulating the Bax/Bcl-2 ratio at the protein level and abnormal ΔΨm, which leads to the suppression of apoptosis.

Background: Epidural steroid injection (ESI) is a common practice for pain treatment since 1953. In 2014, the FDA issued a warning about ESI. Studies have focused on the effect of the particle size and their ability to generate harmful aggregates. Although steroid aggregates provide longer times for reabsorption, therefore a longer anti-inflammatory effect, they are potentially harmful to the central nervous system via embolic mechanisms.Previous studies have established that steroidal aggregates with asizes over 100 mu m are potentially able to occlude blood vessels. Studies by Tiso et al and Benzon et al addressed the role of steroids on CNS adverse events, with similar outcomes. The main difference was on the role of aggregates with a size over 100 mu m, which Benzon et al. attributed to the ability of certain steroid preparations to rapidly precipitate and form large aggregates., Objectives: Studying the effect of the time elapsed between mixing the steroid preparation and injection on the number and size of aggregates with sizes above 100 mu m., Study Design: Original study in basic science., Setting: Basic scienceMETHODS: Steroids evaluated are commonly used in Spain for ESI: betamethasone, triamcinolone, and dexamethasone. The size and number of the aggregates was determined for undiluted commercial steroid preparations in the usual amount for a single and double dosage used for ESI.Samples were examined with a Leica TCS-SP2 microscope at the first, the fifth and the 30th minute after shaking the preparations. Aggregates observed in the different preparations were manually counted and grouped in the following size range: 0-20, 20-50, 50-100, 100-300, 300-500 and > 500 mu m.Statistical analysis was carried out using the R software. Nonparametric techniques were used in the comparison of aggregate size. Global comparison of the groups using the Kruskal-Wallis test and post-hoc comparisons using the Wilcoxon test, adjusting P-values by the Holm method for multiple comparisonsRESULTS: Aggregates present in triamcinolone and betamethasone samples were statistically larger than in dexamethasone samples. Triamcinolone suspensions produced significantly larger aggregates than betamethasone five minutes after mixing. Triamcinolone preparations produced greater particle aggregates (> 500 mu m), which were not present in dexamethasone and betamethasone preparations., Limitations: Study how the human internal factors like blood elements and spinal fluid could interact with steroids and influence the size of the aggregates formed., Conclusions: This study demonstrates that the size of the particles injected depends on the type of steroid and the time allowed between mixing and injecting. The results demonstrate that waiting longer than 5 minutes between mixing and injecting can predispose the formation of potentially harmful aggregates in triamcinolone and betamethasone samples. The presence of greater particle aggregates (> 500 mu m) may occlude some important vessels and arteries with serious adverse results. Vigorous shaking of the injectable could prevent such events., Key Words: Epidural steroid injection, triamcinolone, betamethasone, dexamethasone, steroid aggregates.

Objective: Compounded oral solutions for respiratory illnesses such as the common cold and cough are commonly prepared and dispensed by licensed pharmacists in the United States and Puerto Rico (PR). Standard protocols for their preparation and quality assessment and for patient counseling are available for most of the prescribed compounded solutions. However, in PR there is a common prescription approach colloquially referred to as "mezclitas": mixtures of antitussives, expectorants, decongestants, and other active ingredients available in commercial solutions for which there are no science-driven compounding guidelines for local pharmacists., Methods: This study evaluated the physicochemical stability of a commonly dispensed compounded preparation (containing guaifenesin, dextromethorphan, and dexamethasone) that is used for the treatment of respiratory illnesses in PR. The stability indicators tested included clarity, odor, pH, and viscosity. Changes in stability indicators were evaluated for different storage conditions (ambient temperature and refrigerated) over a period of 6 months., Results: The samples exhibited small changes in color, odor, and viscosity. Although the observed changes were small, they may be indicative of chemical and/or physical transformations that occurred over time. A survey of local pharmacists also evidenced the absence of standardized protocols for the preparation and dispensation of the mezclitas in PR., Conclusion: In spite of the absence of protocols for compounding oral solutions for respiratory illnesses, our study suggests that the stability of such solutions is not heavily compromised. However further chemical and physical testing is needed and the findings of such testing used to develop standardized protocols for the compounding of oral solutions for respiratory illnesses.

Synthetic glucocorticoids are generally preferred over their natural counterparts as these compounds exhibit improved anti-inflammatory potency and glucocorticoid receptor selectivity. However, the biotechnological production of these molecules is often subject to limitations inferred by restricted enzyme stability, selectivity or inhibition thereof. The latter is particularly important during 6α-methylprednisolone production, as the essential C21-hydroxylation of its precursor medrane appears to be hampered by product inhibition of the steroid-21-hydroxylase (CYP21A2). To circumvent this bottleneck, we established a two-step reaction for controlled mixed-culture fermentation, using recombinant E. coli. This process comprises the previously reported C21-hydroxylation of medrane by CYP21A2, followed by an instant derivatization of the hydroxylated product premedrol by chloramphenicol acetyl transferase 1 (CAT1). The CAT1-mediated C21-acetylation prevents the product from regaining access to the enzyme's active site which effectively shifts the chemical equilibrium toward premedrol formation. The successful circumvention of product inhibition at optimized conditions resulted in the formation of more than 1.5 g of product per liter which corresponds to an increase by more than 100 %. Taken together, we demonstrate an efficient system to enhance cytochrome P450-mediated biotransformations, holding great ecologic and economic potential to be applied in industrial processes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Recurrence of glomerulonephritis is the third-leading cause of allograft loss. Graft loss due to IgA nephropathy occurs in 10% at 10-year follow-up. The NEFIGAN trial demonstrated that Target Release Formulation (TRF) of budesonide is a specific treatment for IgA nephropathy targeting intestinal mucosal immunity upstream of disease manifestation with favorable safety profile. We are reporting a case of successful treatment of a patient with posttransplant IgA nephropathy with TRF of budesonide.

The threat of predation can cause increased adrenal secretion of glucocorticoids that, if sustained, can result in chronic stress that might predispose animals to disease, reduced growth, or poor fertility. Fecal glucocorticoid metabolite (FGM) concentrations were measured between September 2011 and July 2012 in two Montana bison ( Bison bison ) herds, one herd living with and the other without wolves ( Canis lupus ) in their habitat. The relevance of FGM concentrations (as measured by the corticosterone enzyme immunoassay) and a confirmation of an acute adrenal response was demonstrated by transient increases ( P <0.001) in FGM concentrations in bison herded through a chute system and following a wolf encounter. The FGM concentrations were higher in the herd with wolves than the herd without wolves overall ( P =0.045), but the monthly differences between the herds were inconsistent (magnitude=0.3-3.5 µg/g; P =0.011 month×herd). The herd with wolves had higher FGM in April, June, July, September, and November, but the herd without wolves had higher FGM in January, February, and May. Seasonal changes in FGM concentrations ( P <0.001) were measured across the year in both herds (magnitude=10.8 and 6.9 µg/g, respectively), exceeding any differences between herds potentially attributable to wolf presence. Concentrations of FGM were highest in April-July and were lowest in January-February. Evidence of a chronic stress response was not found, and increased disease susceptibility in the bison living with wolves seems unlikely.

The glucocorticoid resistance hereditary condition may emerge from the occurrence of point mutations in the glucocorticoid receptor (GR), which could impair its functionality. Because the main feature of such pathology is the resistance of the hypothalamic-pituitary-adrenal axis to the hormone cortisol, we used the GR ligand binding domain three-dimensional structure to perform computational analysis for eight variants known to cause this clinical condition (I559 N, V571A, D641V, G679S, F737L, I747 M, L753F and L773P), aiming to understand, on the atom scale, how they cause glucocorticoid resistance. We observed that the mutations generated a reduced affinity to cortisol and they alter some loop conformations, which could be a consequence from changes in protein motion, which in turn could result from the reduced stability of mutant GR structures. Therefore, the analyzed mutations compromise the GR ligand binding domain structure and cortisol binding, which could characterize the glucocorticoid resistance phenotype., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Clinical application of inhaled glucocorticoids (GCs) has been hampered in the case of steroid-resistant severe asthma. To overcome this limitation, we have developed a series of highly potent GCs, including VSGC12, VSG158, and VSG159 based on the structural insight into the glucocorticoid receptor (GR). Particularly, VSG158 exhibits a maximal repression of lung inflammation and is 10 times more potent than the currently most potent clinical GC, Fluticasone Furoate (FF), in a murine model of asthma. More importantly, VSG158 displays a unique property to reduce neutrophilic inflammation in a steroid-resistant airway inflammation model, which is refractory to clinically available GCs, including dexamethasone and FF. VSG158 and VSG159 are able to deliver effective treatments with reduced off-target and side effects. In addition, these GCs also display pharmacokinetic properties that are suitable for the inhalation delivery method for asthma treatment. Taken together, the excellent therapeutic and side-effect profile of these highly potent GCs holds promise for treating steroid-resistant severe asthma., Competing Interests: Conflict of interest statement: F.S., Z.W., and Z.X. are employees and H.E.X. is a consultant of Palo Alto Pharmaceutics Inc.

The FK506-binding protein 51 (FKBP51) has emerged as a key regulator of endocrine stress responses in mammals and as a potential therapeutic target for stress-related disorders (depression, post-traumatic stress disorder), metabolic disorders (obesity and diabetes) and chronic pain. Recently, FKBP51 has been implicated in several cellular pathways and numerous interacting protein partners have been reported. However, no consensus on the underlying molecular mechanisms has yet emerged. Here, we review the protein interaction partners reported for FKBP51, the proposed pathways involved, their relevance to FKBP51's physiological function(s), the interplay with other FKBPs, and implications for the development of FKBP51-directed drugs., Competing Interests: The authors declare no conflicts of interest.

The glucocorticoid receptors (GR) are members of the nuclear receptor superfamily that regulate growth, development, and many of the biological functions, including metabolism and inflammation, in a ligand dependent behavior. Thus, GRs are vital as therapeutic targets with steroid hormones and steroidal analogues, especially including the glucocorticoids. Studying the molecular mechanism of binding between GR and ligands is fundamentally important to develop applications in the pharmacological industry. The present study was carried out via molecular docking and molecular dynamic (MD) simulations of three GR-ligand complexes formed between the ligand binding domain (LBD) of GR with cortisol (a natural steroid), dexamethasone (a well-known synthetic steroid drug), and chonemorphine (a steroid virtually screened from the "Sri Lankan Flora" web-based information system). The investigation was mainly carried out in terms of macroscopic properties of the ligand-protein interactions and conformational fluctuations of the protein. The results indicated greater stability and a similar behavior of the GR protein in the chonemorphine-GR complex, compared to the other two complexes, GR-dexamethasone and GR-cortisol, in an aqueous medium. The integrity of the protein-substrate complexes was preserved by strong hydrogen bonds formed between the amino acid residues of the binding site of the proteins and ligands. The findings revealed that chonemorphine is a promising agonist to GR and may produce a pharmacological effect like that produced by glucocorticoids. Thus, the obtained knowledge could lead to further investigations of the pharmaceutical potential of chonemorphine and biological functions of GR in vivo.

Glucocorticoids (GCs), acting via the glucocorticoid receptor (GRα), remain the mainstay therapeutic choice for the treatment of inflammation. However, chronic GC use, aside from generating undesirable side-effects, results in GRα down-regulation, often coupled to a decrease in GC-responsiveness, which may culminate in acquired GC resistance. The current study presents evidence for a novel role of the dimerization state of the GRα in mediating GC-mediated GRα turnover. Through comparing the effects of dimerization promoting GCs on down-regulation of a transfected human wild type GRα (hGRwt) or a dimerization deficient GRα mutant (hGRdim), we established that a loss of receptor dimerization restricts GRα turnover, which was supported by the use of the dimerization abrogating Compound A (CpdA), in cells containing endogenous GRα. Moreover, we showed that the dimerization state of the GRα influenced the post-translational processing of the receptor, specifically hyper-phosphorylation at Ser404, which influenced the interaction of GRα with the E3 ligase, FBXW7α, thus hampering receptor turnover via the proteasome. Lastly, the restorative effects of CpdA on the GRα pool, in the presence of Dex, were demonstrated in a combinatorial treatment protocol. These results expand our understanding of factors that contribute to GC-resistance and may be exploited clinically.

The determination of urinary tetrahydroglucocorticoid (THGC) glucuronides might prove helpful in the diagnosis, pathophysiological analysis and assessment of the therapeutic efficacy of the diseases caused by abnormal cortisol secretion. We developed and validated a method for the determination of the THGC glucuronides in human urine using liquid chromatography/electrospray ionization (ESI)-tandem mass spectrometry not requiring enzymatic hydrolysis. The method employed a derivatization using an ESI-enhancing reagent for carboxylic acids, 1-[(4-dimethylaminophenyl)carbonyl]piperazine (DAPPZ), and its isotopologue, 2 H 4 -DAPPZ. The deproteinized urine samples were derivatized with DAPPZ. The 2 H 4 -DAPPZ-derivatized standards of known amounts were then added to the DAPPZ-derivatized urine samples and served as the internal standards. The DAPPZ-derivatization enhanced the assay sensitivity and reduced the sample volume, and the use of 2 H 4 -DAPPZ significantly improved the assay accuracy. The developed method enabled the separate quantification and profiling of the urinary THGC glucuronides and had a satisfactory application for the real sample analysis.

Purpose: The formulation of topical ophthalmic products with appropriate therapeutic effect and patient compliance is a major challenge. To increase the efficiency of the ocular delivery of the drug, the enhancement of water solubility and the contact time of the drug on the surface of the cornea are necessary. In this work, prednisolone (PR)-containing eye drops were formulated with antimicrobial, mucoadhesive biopolymer and PR-cyclodextrin inclusion complex. This approach can be used for the development of innovative ophthalmic formulations., Materials and Methods: After adjusting the optimal physiological parameters, the amount of the required cyclodextrin for the highest penetration of PR was determined by dialysis membrane diffusion study. The viscosity, surface tension and mucoadhesion of the eye drops were measured. The microbiological effectiveness of zinc-hyaluronate (ZnHA) was investigated by a standard method of the European Pharmacopoeia., Results: In this case, no significant difference of surface tension was measured in products with different amounts of cyclodextrin. According to the results of the tensile test, ZnHA as a mucoadhesive biopolymer improves the mucoadhesion of ophthalmic products. The antimicrobial stability of formulations preserved by ZnHA meets requirement B of the European Pharmacopoeia., Conclusion: It can be stated that the innovative PR-containing compositions are suitable for producing mucoadhesive, properly preserved aqueous ophthalmic solutions with increased bioavailability attributes., Competing Interests: Disclosure The author reports no conflicts of interest in this work.

Glucosylation of the 21-hydroxyl group of glucocorticoid changes its solubility into hydrophilicity from hydrophobicity and, as with glucocorticoid glucuronides as a moving object in vivo, it is conceivable that it exhibits the same behavior. Therefore, glucosylation to the 21-hydroxyl group while maintaining the 11 β -hydroxyl group is particularly important, and glucosylation of corticosterone was confirmed by high-resolution mass spectrometry and 1D (¹H and 13 C) and 2D (COSY, ROESY, HSQC-DEPT and HMBC) NMR. Moreover, the difference in bioactivity between corticosterone and corticosterone 21-glucoside was investigated in vitro. Corticosterone 21-glucoside showed greater neuroprotective effects against H₂O₂-induced cell death and reactive oxygen species (ROS) compared with corticosterone. These results for the first time demonstrate that bioconversion of corticosterone through the region-selective glucosylation of a novel compound can present structural potential for developing new neuroprotective agents.

Glucocorticoids (GCs) are a cornerstone in the treatment of lymphoid malignancies such as multiple myeloma (MM) and acute lymphoblastic leukemia (ALL). Yet, prolonged GC use is hampered by deleterious GC-related side effects and the emergence of GC resistance. To tackle and overcome these GC-related problems, the applicability of selective glucocorticoid receptor agonists and modulators was studied, in search of fewer side-effects and at least equal therapeutic efficacy as classic GCs. Compound A (CpdA) is a prototypical example of such a selective glucocorticoid receptor modulator and does not support GR-mediated transactivation. Here, we examined whether the combination of CpdA with the classic GC dexamethasone (Dex) may improve GC responsiveness of MM and ALL cell lines. We find that the combination of Dex and CpdA does not substantially enhance GC-mediated cell killing. In line, several apoptosis hallmarks, such as caspase 3/7 activity, PARP cleavage and the levels of cleaved-caspase 3 remain unchanged upon combining Dex with CpdA. Moreover, we monitor no additional inhibition of cell proliferation and the homologous downregulation of GR is not counteracted by the combination of Dex and CpdA. In addition, CpdA is unable to modulate Dex-liganded GR transactivation and transrepression, yet, Dex-mediated transrepression is also aberrant in these lymphoid cell lines. Together, transrepression-favoring compounds, alone or combined with GCs, do not seem a valid strategy in the treatment of lymphoid malignancies.

Transcriptome-wide time series expression profiling is used to characterize the cellular response to environmental perturbations. The first step to analyzing transcriptional response data is often to cluster genes with similar responses. Here, we present a nonparametric model-based method, Dirichlet process Gaussian process mixture model (DPGP), which jointly models data clusters with a Dirichlet process and temporal dependencies with Gaussian processes. We demonstrate the accuracy of DPGP in comparison to state-of-the-art approaches using hundreds of simulated data sets. To further test our method, we apply DPGP to published microarray data from a microbial model organism exposed to stress and to novel RNA-seq data from a human cell line exposed to the glucocorticoid dexamethasone. We validate our clusters by examining local transcription factor binding and histone modifications. Our results demonstrate that jointly modeling cluster number and temporal dependencies can reveal shared regulatory mechanisms. DPGP software is freely available online at https://github.com/PrincetonUniversity/DP&#95;GP&#95;cluster.

The GOLD 2017 strategy document recommends that the pharmacological management of COPD patients be based on the risk of future exacerbations and the severity of symptoms. A threshold of two moderate exacerbations or one hospitalization is used to define high-risk patients. The FORWARD study was a randomized, double-blind, parallel-group trial that compared 48 weeks' treatment with extrafine beclomethasone dipropionate plus formoterol fumarate (BDP-FF) versus FF in severe COPD patients with a history of one or more exacerbations in the previous year. The new GOLD 2017 recommendations mean that many patients in the FORWARD study are now reclassified as GOLD B. We conducted a post hoc analysis of the FORWARD study, in order to investigate the effects of extrafine BDP/FF in patients with one exacerbation in the previous year, focusing on those categorized as group B using the GOLD 2017 definition. The analysis showed a 35% reduction in exacerbation rate with an inhaled corticosteroid (ICS) + long-acting β-agonist (LABA) versus LABA. We propose that ICS-LABA treatment is a therapeutic option for COPD patients with one exacerbation in the previous year.

The advent of nanomedicine requires novel delivery vehicles to actively target their site of action. Here, we demonstrate the development of lung-targeting drug-loaded liposomes and their efficacy, specificity and safety. Our study focuses on glucocorticoids methylprednisolone (MPS), a commonly used drug to treat lung injuries. The steroidal molecule was loaded into functionalized nano-sterically stabilized unilamellar liposomes (NSSLs). Targeting functionality was performed through conjugation of surfactant protein A (SPANb) nanobodies to form MPS-NSSLs-SPANb. MPS-NSSLs-SPANb exhibited good size distribution, morphology, and encapsulation efficiency. Animal experiments demonstrated the high specificity of MPS-NSSLs-SPANb to the lung. Treatment with MPS-NSSLs-SPANb reduced the levels of TNF-α, IL-8, and TGF-β1 in rat bronchoalveolar lavage fluid and the expression of NK-κB in the lung tissues, thereby alleviating lung injuries and increasing rat survival. The nanobody functionalized nanoparticles demonstrate superior performance to treat lung injury when compared to that of antibody functionalized systems.

Chronic inflammation has been proposed to contribute to the pathogenesis of diet-induced obesity. However, scarce therapeutic options are available to treat obesity and the associated immunometabolic complications. Glucocorticoids are routinely employed for the management of inflammatory diseases, but their pleiotropic nature leads to detrimental metabolic side effects. We developed a glucagon-like peptide-1 (GLP-1)-dexamethasone co-agonist in which GLP-1 selectively delivers dexamethasone to GLP-1 receptor-expressing cells. GLP-1-dexamethasone lowers body weight up to 25% in obese mice by targeting the hypothalamic control of feeding and by increasing energy expenditure. This strategy reverses hypothalamic and systemic inflammation while improving glucose tolerance and insulin sensitivity. The selective preference for GLP-1 receptor bypasses deleterious effects of dexamethasone on glucose handling, bone integrity, and hypothalamus-pituitary-adrenal axis activity. Thus, GLP-1-directed glucocorticoid pharmacology represents a safe and efficacious therapy option for diet-induced immunometabolic derangements and the resulting obesity., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Carbonyl Reductase 1 (CBR1) is a ubiquitously expressed cytosolic enzyme important in exogenous drug metabolism but the physiological function of which is unknown. Here, we describe a role for CBR1 in metabolism of glucocorticoids. CBR1 catalyzes the NADPH- dependent production of 20β-dihydrocortisol (20β-DHF) from cortisol. CBR1 provides the major route of cortisol metabolism in horses and is up-regulated in adipose tissue in obesity in horses, humans and mice. We demonstrate that 20β-DHF is a weak endogenous agonist of the human glucocorticoid receptor (GR). Pharmacological inhibition of CBR1 in diet-induced obesity in mice results in more marked glucose intolerance with evidence for enhanced hepatic GR signaling. These findings suggest that CBR1 generating 20β-dihydrocortisol is a novel pathway modulating GR activation and providing enzymatic protection against excessive GR activation in obesity.

Synthetic glucocorticoids have been detected in environmental waters and their biological potency have raised concerns of their impact on aquatic vertebrates especially fish. In this study, developing zebrafish larvae exposed to representative glucocorticoids (dexamethasone, prednisolone and triamcinolone) at 50 pM to 50 nM from 3 h post-fertilisation to 5 days post-fertilisation were investigated. Microarray analysis identified 1255, 1531, and 2380 gene probes, which correspondingly mapped to 660, 882 and 1238 human/rodent homologs, as deregulated by dexamethasone, prednisolone and triamcinolone, respectively. A total of 248 gene probes which mapped to 159 human/rodent homologs were commonly deregulated by the three glucocorticoids. These homologs were associated with over 20 molecular functions from cell cycle to cellular metabolisms, and were involved in the development and function of connective tissue, nervous, haematological, and digestive systems. Glucocorticoid receptor signalling, NRF2-mediated oxidative stress response and RAR signalling were among the top perturbed canonical pathways. Morphological analyses using four transgenic zebrafish lines revealed that the hepatic and endothelial-vascular systems were affected by all three glucocorticoids while nervous, pancreatic and myeloid cell systems were affected by one of them. Quantitative real-time PCR detected significant change in the expression of seven genes at 50 pM of all three glucocorticoids, a concentration comparable to total glucocorticoids reported in environmental waters. Three genes (cry2b, fbxo32, and klhl38b) responded robustly to all glucocorticoid concentrations tested. The common deregulated genes with the associated biological processes and morphological changes can be used for biological inference of glucocorticoid exposure in fish for future studies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: Glucocorticoids, widely used in the perinatal period, may be associated with adverse neurodevelopmental effects., Objectives: To analyze neurodevelopmental outcomes in a cohort of very low birth weight newborns treated with antenatal and/or postnatal corticosteroids., Materials and Methods: This was a prospective cohort study in which we included all very low birth weight babies admitted to the neonatal intensive care unit of a tertiary hospital between 2008 and 2013. We compared the neurodevelopment among very low birth-weight newborns who did not receive prenatal corticosteroid therapy and those who received a complete course (two doses of 12 mg betamethasone) and an incomplete course (one dose), and between those who did not receive postnatal corticosteroid therapy and those who received it (systemic dexamethasone after the first week of life). Motor, neurosensory and cognitive functions, as well as behavior disorders during the first two years of age were evaluated., Results: A total of 225 very low birth weight newborns were analyzed; 83.6% received prenatal corticosteroid therapy (24% incomplete treatment schedule and 59.6% complete schedule). Only 13.3% received systemic dexamethasone during the postnatal period. Neurological monitoring was performed corticosteroid therapy and more neurological disorders, and no significant differences were found among those who received complete and incomplete courses of prenatal corticosteroid therapy., Conclusion: These results did not demonstrate an association between perinatal corticosteroid therapy and worse neurodevelopmental outcomes in very low birth weight newborns.

An efficient and inexpensive method using vortex-assisted surfactant-enhanced emulsification microextraction (VASEME) based on solidification of floating organic droplet coupled with ultraperformance liquid chromatography-tandem mass spectrometry is proposed for the analysis of glucocorticoids in water samples (river water and hospital wastewater). VASEME was optimized by the experimental validation of Plackett-Burman design and central composite design, which has been co-related to experimental design. Plackett-Burman design showed that factors such as vortex time, surfactant concentration, and pH significantly affect the extraction efficiency of the method. Method validation was characterized by an acceptable calibration range of 1-1000 ng L -1 , and the limit of detection was in the range from 2.20 to 8.12 ng L -1 for glucocorticoids. The proposed method was applied to determine glucocorticoids in river water and hospital wastewater in Lucknow, India. It is reliable and rapid and has potential application for analysis of glucocorticoids in environmental aqueous samples. Graphical Abstract Low density based extraction of gluococorticoids by using design of experiment.

Human milk (HM) contains a complex array of hormones, including members of the glucocorticoid family. The predominant glucocorticoids, cortisol and cortisone may influence the growth and behaviour of the breastfed infant. However, little is understood of the factors regulating the levels of these hormones within HM. The aim of the study was to examine HM cortisol and cortisone concentration, measured in samples collected at each feed during a 24 hour period. Twenty three exclusively breastfeeding mothers collected milk, prior to and after each breastfeeding session over 24 hour period at 3.2(1.60) months. HM cortisol and cortisone levels were measured using high pressure liquid chromatography mass spectroscopy. Cortisone was the predominant glucocorticoid (3.40 ng/ml), and cortisol was detected in all samples (1.62 ng/ml). A positive correlation was found between cortisone and cortisol (r = 0.61, y = 1.93 ± 0.24, p < 0.0001). Cortisol and cortisone concentrations were significantly higher in feeds in the morning (2.97 ng/ml and 4.88 ng/ml), compared to afternoon (1.20 ng/ml and 3.54 ng/ml), evening (0.69 ng/ml and 2.13 ng/ml) and night (1.59 and 3.27 ng/ml). No difference was found between glucocorticoids level of the milk expressed for collection either before or immediately after the breastfeed, or between milk collected from the left or right breast. This study shows that HM glucocorticoid concentrations exhibit a 24 hour pattern, with highest peak levels in the early morning, reflecting the circadian pattern as previously reported in plasma. Thus, HM glucocorticoid concentrations are likely to reflect those in the maternal circulation.

Background: Numbers of giraffes are declining rapidly in their native habitat. As giraffe research and conservation efforts increase, the demand for more complete measures of the impact of conservation interventions and the effects of captive environments on animal health and welfare have risen. We compared the ability of six different enzyme immunoassays to quantify changes in fecal glucocorticoid metabolites (FGM) resulting from three sources: adrenocorticotropic hormone stimulation test, transport, and time of day that samples were collected., Results: Two male giraffes underwent ACTH injections; all six assays detected FGM increases following injection for Giraffe 1, while only three assays detected FGM increases following injection for Giraffe 2. Consistent with other ruminant species, the two 11-oxoetiocholanolone assays (one for 11,17-dioxoandrostanes and the other for 3α,11-oxo metabolites) measured the most pronounced and prolonged elevation of FGM, while an assay for 3β,11β-diol detected peaks of smaller magnitude and duration. Both of the 11-oxoetiocholanolone assays detected significant FGM increases after transport in Giraffes 3-7, and preliminary data suggest FGM detected by the assay for 11,17-dioxoandrostanes may differ across time of day., Conclusions: We conclude the assay for 11,17-dioxoandrostanes is the most sensitive assay tested for FGM in giraffes and the assay for FGM with a 5β-3α-ol-11-one structure is also effective. 11-oxoetiocholanolone enzyme immunoassays have now been demonstrated to be successful in a wide variety of ruminant species, providing indirect evidence that 5β-reduction may be a common metabolic pathway for glucocorticoids in ruminants. As FGM peaks were detected in at least some giraffes using all assays tested, giraffes appear to excrete a wide variety of different FGM. The assays validated here will provide a valuable tool for research on the health, welfare, and conservation of giraffes.

Dexamethasone (Dex) is an effective glucocorticoid in treating inflammation and preventing rejection reaction. However, the side effects limit its clinical application. To improve its druggable profile, the conjugates of RGD-peptide-modified Dex were presented and their enhanced anti-inflammation activity, minimized osteoporotic action, and nanoscaled assembly were explored. (RGD stands for Arg-Gly-Asp. Standard single letter biochemical abbreviations for amino acids have been used throughout this paper.) In respect of the rejection reaction, the survival time of the implanted myocardium of the mice treated with 1.43 µmol/kg/d of the conjugates for 15 consecutive days was significantly longer than that of the mice treated with 2.5 µmol/kg/d of Dex, and the conjugates, but not Dex, exhibited no toxic action. At a single dose of 14.3 µmol/kg (100 times minimal effective dose, 0.143 µmol/kg), the conjugates induced no liver, kidney, or systemic toxicity. At the dose of 1.43 µmol/kg, the conjugates, but not Dex, prolonged the bleeding time of the mice, and inhibited the thrombosis of the rats. In water and rat plasma, the conjugates formed nanoparticles of 14-250 and 101-166 nm in diameter, respectively. Since the nanoparticles of ~100 nm in size cannot be entrapped by macrophages in the circulation, RGDF-Dex would particularly be worthy of development, since its nanoparticle diameter is 101 nm.

The North Atlantic right whale, Eubalaena glacialis (NARW), a critically endangered species that has been under intensive study for nearly four decades, provides an excellent case study for applying modern methods of conservation physiology to large whales. By combining long-term sighting histories of known individuals with physiological data from newer techniques (e.g., body condition estimated from photographs; endocrine status derived from fecal samples), physiological state and levels of stress can be estimated despite the lack of any method for nonlethal capture of large whales. Since traditional techniques for validating blood assays cannot be used in large whales, assays of fecal hormones have been validated using information on age, sex, and reproductive state derived from an extensive NARW photo-identification catalog. Using this approach, fecal glucocorticoids have been found to vary dramatically with reproductive state. It is therefore essential that glucocorticoid data be interpreted in conjunction with reproductive data. A case study correlating glucocorticoids with chronic noise is presented as an example. Keys to a successful research program for this uncatchable species have included: consistent population monitoring over decades, data-sharing across institutions, an extensive photo-identification catalog that documents individual histories, and consistent efforts at noninvasive collection of samples over years. Future research will require flexibility to adjust to changing distributions of populations., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Due to its cytotoxic effect in lymphoid cells, dexamethasone is widely used in the treatment of multiple myeloma (MM). However, only a subset of myeloma patients responds to high-dose dexamethasone. Despite the undeniable anti-myeloma benefits of dexamethasone, significant adverse effects have been reported. We re-evaluate the anti-tumor effect of dexamethasone according to the molecular heterogeneity of MM. We demonstrated that the pro-death effect of dexamethasone is related to the genetic heterogeneity of MM because sensitive cell lines were restricted to MAF and MMSET signature subgroups, whereas all CCND1 cell lines (n = 10) were resistant to dexamethasone. We demonstrated that the glucocorticoid receptor expression was an important limiting factor for dexamethasone-induced cell death and we found a correlation between glucocorticoid receptor levels and the induction of glucocorticoid-induced leucine zipper (GILZ) under dexamethasone treatment. By silencing GILZ, we next demonstrated that GILZ is necessary for Dex induced apoptosis while triggering an imbalance between anti- and pro-apoptotic Bcl-2 proteins. Finally, the heterogeneity of the dexamethasone response was further confirmed in vivo using myeloma xenograft models. Our findings suggested that the effect of dexamethasone should be re-evaluated within molecular subgroups of myeloma patients to improve its efficacy and reduce its adverse effects.

Glucocorticoids, widely used in inflammatory disorders, rapidly increase bone fragility and, therefore, fracture risk. However, common bone densitometry measurements are not sensitive enough to detect these changes. Moreover, densitometry only partially recognizes treatment-induced fracture reductions in osteoporosis. Here, we tested whether the reference point indentation technique could detect bone tissue property changes early after glucocorticoid treatment initiation. After initial laboratory and bone density measurements, patients were allocated into groups receiving calcium + vitamin D (Ca+D) supplements or anti-osteoporotic drugs (risedronate, denosumab, teriparatide). Reference point indentation was performed on the cortical bone layer of the tibia by a handheld device measuring bone material strength index (BMSi). Bone mineral density was measured by dual-energy X-ray absorptiometry (DXA). Although Ca+D-treated patients exhibited substantial and significant deterioration, risedronate-treated patients exhibited no significant change, and both denosumab- and teriparatide-treated participants exhibited significantly improved BMSi 7 weeks after initial treatment compared with baseline; these trends remained stable for 20 weeks. In contrast, no densitometry changes were observed during this study period. In conclusion, our study is the first to our knowledge to demonstrate that reference point indentation is sensitive enough to reflect changes in cortical bone indentation after treatment with osteoporosis therapies in patients newly exposed to glucocorticoids., (© 2015 American Society for Bone and Mineral Research.)

Background: The QuantiFERON-TB Gold In-Tube (QFT-GIT) is a newly developed but widely used interferon-γ release assay for diagnosing tuberculosis (TB). However, research has not determined whether age or the use of an immune suppressive or anti-TB treatment influences this assay's ability to detect TB. We assessed the QFT-GIT diagnostic performance for active tuberculosis (ATB) in children and adults in an endemic country and explored the effects of glucocorticoids and anti-TB therapy on the diagnostic value of the QFT-GIT., Methods: A total of 60 children and 212 adults with suspected ATB were evaluated with the QFT-GIT. The association between the QFT-GIT diagnostic value and pretreatment factors was qualitatively and quantitatively assessed., Results: The sensitivity of the QFT-GIT was 83.9% (95% CI 66.3%-94.6%) in children, and 73.7% (95% CI 57.8%-85.2%) in adults. Glucocorticoids affected the mitogen-stimulated response in both children and adults. In subjects undergoing glucocorticoid pretreatment, 25.0% of the children presented with false-negative QFT-GIT results, 28.6% of adults presented with indeterminate results. For subjects pre-treated with anti-TB drugs, 44.4% presented with false-negative QFT-GIT results., Conclusions: The QFT-GIT has higher sensitivity and specificity in children than adults. Glucocorticoid treatment negatively impacts the diagnostic value of the QFT-GIT in all age groups. Anti-TB treatment decreases the sensitivity of the QFT-GIT. Therefore, we recommend that the QFT-GIT assay be performed before TB-specific treatment is initiated and the test should not be used on people undergoing immunosuppression treatment, regardless of their age. A quantitative analysis of the QFT-GIT could be useful for assessing and monitoring TB-specific and non-specific immunity during conversion of the disease.

Purpose: To evaluate the safety and efficacy of 1.5% dexamethasone nanoparticle (DexNP) drops in eyes with non-infectious uveitic macular oedema and vitritis., Methods: In a prospective pilot study, DexNP drops were administered four times a day for 4 weeks followed by drops tapering over a period of another 4 weeks. Follow-up time was 12 weeks., Results: Five eyes with macular oedema and three eyes with vitritis were included in the study. Best corrected visual acuity (BCVA) significantly improved from a median of 0.2 logMAR to a median of 0.15 logMAR at 4 weeks' time (p < 0.05). Median BCVA was 0.175 logMAR and 0.2 logMAR, at week 8 and 12, respectively (p > 0.05). Macular oedema significantly improved at all time-points as compared to baseline (p < 0.05) and resolved in all eyes during follow-up. One eye had macular oedema relapse at week 12. Vitritis improved in all eyes and resolved completely in two eyes. One eye had intraocular pressure (IOP) elevation which was well controlled with topical antihypertensive treatment, and one eye had cataract progression., Conclusion: This short pilot study demonstrates favourable effect of 1.5% DexNP eye drops on eyes with non-infectious uveitic macular oedema and vitritis. Further comparative long-term studies are warranted to assess this effect., (© 2015 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Glucocorticoids remain the cornerstone of treatment for inflammatory conditions, but their utility is limited by a plethora of side effects. One of the key goals of immunotherapy across medical disciplines is to minimize patients' glucocorticoid use. Increasing evidence suggests that variations in the adaptive immune response play a critical role in defining the dose of glucocorticoids required to control an individual's disease, and Th17 cells are strong candidate drivers for nonresponsiveness [also called steroid resistance (SR)]. Here we use gene-expression profiling to further characterize the SR phenotype in T cells and show that Th17 cells generated from both SR and steroid-sensitive individuals exhibit restricted genome-wide responses to glucocorticoids in vitro, and that this is independent of glucocorticoid receptor translocation or isoform expression. In addition, we demonstrate, both in transgenic murine T cells in vitro and in an in vivo murine model of autoimmunity, that Th17 cells are reciprocally sensitive to suppression with the calcineurin inhibitor, cyclosporine A. This result was replicated in human Th17 cells in vitro, which were found to have a conversely large genome-wide shift in response to cyclosporine A. These observations suggest that the clinical efficacy of cyclosporine A in the treatment of SR diseases may be because of its selective attenuation of Th17 cells, and also that novel therapeutics, which target either Th17 cells themselves or the effector memory T-helper cell population from which they are derived, would be strong candidates for drug development in the context of SR inflammation.

Background and Purpose: International asthma guidelines recommend that inhaled glucocorticoids be used as a monotherapy in all patients with mild to moderate disease because of their ability to suppress airways inflammation. Current evidence suggests that the therapeutic benefit of glucocorticoids is due to the transactivation and transrepression of anti-inflammatory and pro-inflammatory genes respectively. However, the extent to which clinically relevant glucocorticoids are equivalent in their ability to modulate gene expression is unclear., Experimental Approach: A pharmacodynamics investigation of glucocorticoid receptor (GR)-mediated gene transactivation in BEAS-2B human airway epithelial cells was performed using a glucocorticoid response element luciferase reporter coupled with an analysis of glucocorticoid-inducible genes encoding proteins with anti-inflammatory and adverse-effect potential., Key Results: Using transactivation as a functionally relevant output, a given glucocorticoid displayed a unique, gene expression 'fingerprint' where intrinsic efficacy and GR density were essential determinants. We showed that depending on the gene selected for analysis, a given glucocorticoid can behave as an antagonist, partial agonist, full agonist or even 'super agonist'. In the likely event that different, tissue-dependent gene expression profiles are reproduced in vivo, then the anti-inflammatory and adverse-effect potential of many glucocorticoids currently available as asthma therapeutics may not be equivalent., Conclusions and Implications: The generation of gene expression 'fingerprints' in target and off-target human tissues could assist the rational design of GR agonists with improved therapeutic ratios. This approach could identify compounds that are useful in the management of severe asthma and other inflammatory disorders where systemic exposure is desirable., (© 2014 The British Pharmacological Society.)

To enhance the solubility and in vitro dissolution of fluticasone propionate (FP), a novel approach was developed with mechanochemical treatment. The order of solubilizing effect of β-CD derivatives was observed as HP-β-CD-SBE-β-CD-β-CD-HE-β-CD, consequently, HP-β-CD showed the highest stability constant. To further improve FP solubility, FP and HP-β-CD were grinded using a roll mill, the optimal conditions, determined through single factor experiments, were as follows: rotation frequency of 60 Hz; milling time of 6h. mass ratio of 1: 7. In comparison with pure FP, a 280-fold increase in solubility and a 2.15-fold higher dissolution rate of ground mixture was obtained. The characterization of FP and HP-β-CD complexes had been analyzed by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), powder X-ray diffractometry (PXRD) and fourier transform infrared spectroscopy (FT-IR). The results suggested that the interaction between FP and HP-β-CD was strengthened and an amorphous ground mixture was gained. After stored for 60 days, the ground mixtures were stable both chemically and physically.

The inflammatory response is a highly regulated process, and its dysregulation can lead to the establishment of chronic inflammation and, in some cases, to death. Inflammation is the cause of several diseases, including rheumatoid arthritis, inflammatory bowel diseases, multiple sclerosis, and asthma. The search for agents inhibiting inflammation is a great challenge as the inflammatory response plays an important role in the defense of the host to infections. Marine invertebrates are exceptional sources of new natural products, and among those diterpenoids secondary metabolites exhibit notable anti-inflammatory properties. Novel anti-inflammatory diterpenoids, exclusively produced by marine organisms, have been identified and synthetic molecules based on those structures have been obtained. The anti-inflammatory activity of marine diterpenoids has been attributed to the inhibition of Nuclear Factor-κB activation and to the modulation of arachidonic acid metabolism. However, more research is necessary to describe the mechanisms of action of these secondary metabolites. This review is a compilation of marine diterpenoids, mainly isolated from corals, which have been described as potential anti-inflammatory molecules.

The purpose of this study was to develop a clear aqueous mixed nanomicellar formulation (MNF) of dexamethasone utilizing both D-α-tocopherol polyethylene glycol-1000 succinate (Vit E TPGS) and octoxynol-40 (Oc-40). In this study, Vit E TPGS and Oc-40 are independent variables. Formulations were prepared following solvent evaporation method. A three level full-factorial design was applied to optimize the formulation based on entrapment efficiency, size, and polydispersity index (PDI). A specific blend of Vit E TPGS and Oc-40 at a particular wt% ratio (4.5:2.0) produced excellent drug entrapment, loading, small mixed nanomicellar size and narrow PDI. Solubility of DEX in MNF is improved by ~6.3-fold relative to normal aqueous solubility. Critical micellar concentration (CMC) for blend of polymers (4.5:2.0) was found to be lower (0.012 wt%) than the individual polymers (Vit E TPGS (0.025 wt%) and Oc-40 (0.107 wt%)). No significant effect on mixed nanomicellar size and PDI with one-factor or multi-factor interactions was observed. Qualitative (1)H NMR studies confirmed absence of free drug in the outer aqueous MNF medium. MNF appeared to be highly stable. Cytotoxicity studies on rabbit primary corneal epithelial cells did not indicate any toxicity suggesting MNF of dexamethasone is safe and suitable for human topical ocular drops after further in vivo evaluations.

The impact of formulation variables on aerodynamic and electrostatic properties of dry powder aerosol particles is of great importance to the development of efficient and reproducible inhaler products. Systematic evaluation requires a well-designed series of experiments using appropriate methods. A factorial experimental design was employed. In broad terms, the conditions considered were two drugs, albuterol and budesonide, in combination with different excipients, drug concentrations, delivered doses, and metering system (capsule composition) and sampled under different flow conditions using standard entrainment tubes. Samples were collected in an electrical low-pressure impactor, to evaluate distribution of electrostatic properties, and an Andersen eight-stage nonviable cascade impactor, to estimate aerodynamic particle size distribution, concurrently. The deposition studies allowed calculation of approximate per particle charge levels for drug. The results showed very high particle charge levels, often in the 1,000-10,000 of elementary charges per particle range, orders of magnitude higher than charge levels predicted by the Boltzmann charge distribution. The charge levels are considerably higher than had previously been estimated (200e per particle).

This work reports the preparation of modified poly(vinyl alcohol) PVA decorated with two different hydrophobic side-arms. Each material displays various percentages of pendant oleate and cholesteryl groups. The molar amount and the difference between their respective physico-chemical contributions are able to induce size variations of the formed micelles in aqueous media. These large amphiphilic structures hold a hydrophobic core ready for the incorporation of strong hydrophobic species such as cholesterol or prednisolone, a well-known low-water soluble steroid-type drug. In the presence of a steroid derivative, the modified PVA could form smaller and dense nanoparticles loaded in their cores. The synthesis of each polymer was controlled by (1)H NMR and FT-IR spectroscopies. The size of the empty micelles and the diameter of the loaded nanoparticles were determined by dynamic light scatting (DLS) studies. The measurement of the critical micelle concentration (CMC) was carried out for each polymer sample by fluorescence probing using pyrene. The amount of cholesterol incorporated into the hydrophobic core was estimated and all binding modes between the steroids and each amphiphilic polymer were also discussed in regard to their size distribution, concentration and stability., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Respiratory distress syndrome (RDS) persists as a prevalent cause of infant morbidity and mortality. We have previously demonstrated that deletion of Erk3 results in pulmonary immaturity and neonatal lethality. Using RNA sequencing, we identified corticotrophin releasing hormone (CRH) and surfactant protein B (SFTPB) as potential molecular mediators of Erk3-dependent lung maturation. In this study, we characterized the impact of antenatal glucocorticoids and postnatal surfactant on neonatal survival of Erk3 null mice., Methods: In a double crossover design, we administered dexamethasone (dex) or saline to pregnant dams during the saccular stage of lung development, followed by postnatal surfactant or saline via inhalation intubation. Survival was recorded, and detailed lung histological analysis and staining for CRH and SFTPB protein expression were performed., Results: Without treatment, Erk3 null pups die within 6 h of birth with reduced aerated space, impaired thinning of the alveolar septa, and abundant glycogen stores, as described in human RDS. The administration of dex and surfactant improved RDS-associated lethality of Erk3(-/-) pups and partially restored functional fetal lung maturation by accelerating the downregulation of pulmonary CRH and partially rescuing the production of SFTPB., Conclusion: These findings emphasize that Erk3 is integral to terminal differentiation of type II cells, SFTPB production, and fetal pulmonary maturity.

The evolution of glucocorticoid drugs was driven by the demand of lowering the unwanted side effects, while keeping the beneficial anti-inflammatory effects. Potency is an important aspect of this evolution as many undesirable side effects are associated with use of high-dose glucocorticoids. The side effects can be minimized by highly potent glucocorticoids that achieve the same treatment effects at lower doses. This demand propelled the continuous development of synthetic glucocorticoids with increased potencies, but the structural basis of their potencies is poorly understood. To determine the mechanisms underlying potency, we solved the X-ray structures of the glucocorticoid receptor (GR) ligand-binding domain (LBD) bound to its endogenous ligand, cortisol, which has relatively low potency, and a highly potent synthetic glucocorticoid, mometasone furoate (MF). The cortisol-bound GR LBD revealed that the flexibility of the C1-C2 single bond in the steroid A ring is primarily responsible for the low affinity of cortisol to GR. In contrast, we demonstrate that the very high potency of MF is achieved by its C-17α furoate group completely filling the ligand-binding pocket, thus providing additional anchor contacts for high-affinity binding. A single amino acid in the ligand-binding pocket, Q642, plays a discriminating role in ligand potency between MF and cortisol. Structure-based design led to synthesis of several novel glucocorticoids with much improved potency and efficacy. Together, these results reveal key structural mechanisms of glucocorticoid potency and provide a rational basis for developing novel highly potent glucocorticoids.

Chinese goral (Naemorhedus griseus) are a threatened species in Thailand and the focus of captive breeding for possible reintroduction. However, little is known of their biology or what factors in the captive environment affect welfare. Our objective was to determine the impact of gender, season, and management on goral adrenal activity. We hypothesized that differences in fecal glucocorticoid concentrations would be related to animal density. Fecal samples were collected 3 days/week for 1 year from 63 individuals (n = 32 males, 31 females) at two facilities that house the majority of goral in Thailand: Omkoi Wildlife Sanctuary (Omkoi), an off-exhibit breeding center that houses goral in individual pens (16 pens; n = 8 males, 8 females) and in small family groups (8 pens; n = 8 males, 8 females); and the Chiang Mai Night Safari (NS), a zoo that maintains 31 goral (n = 17 males, 14 females) in one large pen. Glucocorticoid metabolite concentrations were higher in male than female goral at Omkoi throughout the year, and there was a seasonal effect on adrenal activity (p<0.05). Goral at Omkoi and NS were used to test the effect of animal density on fecal glucocorticoid excretion of goral housed in similar-sized enclosures. Overall, the highest levels were found at NS (n = 31 adults/pen; 27 m2 per animal) compared to Omkoi (n = 2 adults/pen; 400 m2 per animal) (p<0.05). Overall findings support our hypothesis that animal density and aspects of the captive environment impact adrenal steroid activity in captive goral. In addition, gender and season also had significant effects on glucocorticoid metabolite production. Potential stressors pertaining to the welfare of this species were identified, which will guide future efforts to improve management and create self-sustaining and healthy populations of this threatened species.

Background: Validation of a method for the minimally-invasive measurement of physiological stress will help understanding of risk factors that may contribute to stress-associated events including recrudescence of Equid herpesvirus (EHV), which is anecdotally associated with sales consignment of pregnant Thoroughbred mares. In this study we compared two similar groups of late-gestation Thoroughbred broodmares on the same farm: a consigned Sales group (N = 8) and a non-consigned Control group (N = 6). The Sales mares were separated from their paddock companions and grouped prior to their preparation for, transport to, and return from the sales venue. Both groups were monitored by sampling at regular intervals from 5 days prior to until 14 days after the sales date (D0) to measure physiological stress in terms of changes in faecal glucocorticoid metabolite (FGM) concentrations, and for event-related viral recrudescence via daily body temperature measurements and periodic nasal swabs for PCR analysis for EHV-1 and -4 DNA., Results: In both groups, FGM levels increased post-sales before returning to pre-sales levels. Specifically, FGM concentrations in the Sales mares were significantly higher on D + 3 and D + 10 than on D-4 and D-3 (F = 12.03, P < 0.0001, Post hoc: P = 0.0003-0.0008) and in the Control group FGM concentrations were higher on D + 10 than D-4 (F = 5.52, P = 0.004, Post hoc: P = 0.005). Interestingly, mean FGM levels in Control mares were significantly higher at 4 of the 5 sampling points (t = 5.64-2.25, p = 0.0001-0.044). Only one (Sales) mare showed PCR evidence of EHV-1 shedding., Conclusions: Using FGM to measure physiological stress was supported by the increases observed in all mares after Sales consignment, including those not consigned to the sale. Monitoring FGM levels therefore represents an appropriate, minimally-invasive method for future studies to assess the contribution of physiological stress to EHV recrudescence in horses transported to sales or equestrian events.

Glucocorticoid (GC) hormones have been introduced as therapeutic agents in blood cancers six decades ago. The effectiveness of GC treatment stems from its ability to induce apoptotic death of hemopoietic cells. A major impediment in GC therapy is the acquisition of resistance to the drug upon repeated treatment. In addition, some blood cancers are a priori resistant to GC therapy. Usually, resistance to GC correlates with poor prognosis. Albeit the wide use of GC in clinical practice, their mode of action is not fully understood. The cellular response to GC is initiated by its binding to the cytosolic GC receptor (GR) that translocates to the nucleus and modulates gene expression. However, nuclear activities of GR occur in both apoptosis-sensitive and apoptosis-resistant cells. These apparent controversies can be resolved by deciphering non-genomic effects of GCs and the mode by which they modulate the apoptotic response. We suggest that non-genomic consequences of GC stimulation determine the cell fate toward survival or death. Understanding the cellular mechanisms of GC apoptotic sensitivity contributes to the development of new modalities for overcoming GC resistance.