How an appreciation of dynamics has altered our understanding of the HPA axis.

Rhythmicity is a intrinsic feature of biological systems, including the hypothalamic-pituitary-adrenal axis, a mammalian neurohormonal system crucial both in daily life and as a network that responds to stressful stimuli. Circadian and ultradian rhythmicity underlie HPA activity in rodents and in humans, regulating gene expression, metabolism and behavior, and adverse consequences occur when rhythms are disturbed. In the assessment of human disease, the complexity of HPA rhythmicity is rarely acknowledged or understood, and is currently a limitation to better diagnosis and treatment. However, the recent emergence of ambulatory, high frequency and blood-free hormone sampling techniques has the promise to substantially change our understanding of the function of HPA axis in healthy normal life, and provide new opportunities for the diagnosis and treatment of disease.

Ultradian hydrocortisone replacement alters neuronal processing, emotional ambiguity, affect and fatigue in adrenal insufficiency: The PULSES trial.

BACKGROUND: Primary adrenal insufficiency (PAI) mortality and morbidity remain unacceptably high, possibly arising as glucocorticoid replacement does not replicate natural physiology. A pulsatile subcutaneous pump can closely replicate cortisol's circadian and ultradian rhythm. OBJECTIVES: To assess the effect of pump therapy on quality of life, mood, functional neuroimaging, behavioural/cognitive responses, sleep and metabolism. METHODS: A 6-week randomised, crossover, double-blinded and placebo-controlled feasibility study of usual dose hydrocortisone in PAI administered as either pulsed subcutaneous or standard care in Bristol, United Kingdom (ISRCTN67193733). Participants were stratified by adrenal insufficiency type. All participants who received study drugs are included in the analysis. The primary outcome, the facial expression recognition task (FERT), occurred at week 6. RESULTS: Between December 2014 and 2017, 22 participants were recruited - 20 completed both arms, and 21 were analysed. The pump was well-tolerated. No change was seen in the FERT primary outcome; however, there were subjective improvements in fatigue and mood. Additionally, functional magnetic resonance imaging revealed differential neural processing to emotional cues and visual stimulation. Region of interest analysis identified the left amygdala and insula, key glucocorticoid-sensitive regions involved in emotional ambiguity. FERT post hoc analysis confirmed this response. There were four serious adverse events (AE): three intercurrent illnesses requiring hospitalisation (1/3, 33.3% pump) and a planned procedure (1/1, 100% pump). There was a small number of expected AEs: infusion site bruising/itching (3/5, 60% pump), intercurrent illness requiring extra (3/7, 42% pump) and no extra (4/6, 66% pump) steroid. CONCLUSIONS: These findings support the administration of hormone therapy that mimics physiology.

High-resolution daily profiles of tissue adrenal steroids by portable automated collection.

Rhythms are intrinsic to endocrine systems, and disruption of these hormone oscillations occurs at very early stages of the disease. Because adrenal hormones are secreted with both circadian and ultradian periods, conventional single-time point measurements provide limited information about rhythmicity and, crucially, do not provide information during sleep, when many hormones fluctuate from nadir to peak concentrations. If blood sampling is attempted overnight, then this necessitates admission to a clinical research unit, can be stressful, and disturbs sleep. To overcome this problem and to measure free hormones within their target tissues, we used microdialysis, an ambulatory fraction collector, and liquid chromatography-tandem mass spectrometry to obtain high-resolution profiles of tissue adrenal steroids over 24 hours in 214 healthy volunteers. For validation, we compared tissue against plasma measurements in a further seven healthy volunteers. Sample collection from subcutaneous tissue was safe, well tolerated, and allowed most normal activities to continue. In addition to cortisol, we identified daily and ultradian variation in free cortisone, corticosterone, 18-hydroxycortisol, aldosterone, tetrahydrocortisol and allo-tetrahydrocortisol, and the presence of dehydroepiandrosterone sulfate. We used mathematical and computational methods to quantify the interindividual variability of hormones at different times of the day and develop "dynamic markers" of normality in healthy individuals stratified by sex, age, and body mass index. Our results provide insight into the dynamics of adrenal steroids in tissue in real-world settings and may serve as a normative reference for biomarkers of endocrine disorders (ULTRADIAN, NCT02934399).

Challenges and solutions to recruitment of neonates and children having cardiac surgery into a study using a novel sampling device.

OBJECTIVE: To narratively describe the challenges and solutions required in delivering a non-commercial study of children undergoing cardiac surgery using a novel subcutaneous hormone collection device. RESULTS: The challenges faced by the research team are divided into those of conducting healthcare research in children and those specific to this study. Many of the issues of conducting healthcare research in children can and have been overcome by structural and institutional culture change-normalising and embedding research as part of good clinical care. The issues specific to insertion and maintenance of the novel collection device can be overcome by education and support of the clinical teams. The increased incentives and resources of commercial research may have overcome many of these.

Modelling the dynamic interaction of systemic inflammation and the hypothalamic-pituitary-adrenal (HPA) axis during and after cardiac surgery.

Major surgery and critical illness produce a potentially life-threatening systemic inflammatory response. The hypothalamic-pituitary-adrenal (HPA) axis is one of the key physiological systems that counterbalances this systemic inflammation through changes in adrenocorticotrophic hormone (ACTH) and cortisol. These hormones normally exhibit highly correlated ultradian pulsatility with an amplitude modulated by circadian processes. However, these dynamics are disrupted by major surgery and critical illness. In this work, we characterize the inflammatory, ACTH and cortisol responses of patients undergoing cardiac surgery and show that the HPA axis response can be classified into one of three phenotypes: single-pulse, two-pulse and multiple-pulse dynamics. We develop a mathematical model of cortisol secretion and metabolism that predicts the physiological mechanisms responsible for these different phenotypes. We show that the effects of inflammatory mediators are important only in the single-pulse pattern in which normal pulsatility is lost-suggesting that this phenotype could be indicative of the greatest inflammatory response. Investigating whether and how these phenotypes are correlated with clinical outcomes will be critical to patient prognosis and designing interventions to improve recovery.

An Investigation of Pulse Oximetry Levels During Swallowing in Healthy Adults and in Individuals with Severe and Very Severe Chronic Obstructive Pulmonary Disease.

To compare pulse oximetry (PO) levels during swallowing in healthy adults and adults with severe chronic obstructive pulmonary disease (COPD). Participants included two groups of 60 healthy adults, balanced for gender. The young group ranged from 18 to 38 years, and the older group from 60 to 87 years. In addition, there were 11 participants with COPD aged 43 to 82 years. PO levels were collected as each participant swallowed 10 mL of water, 10 mL of applesauce, and a piece of diced pear (three trials each). Analyses for the healthy groups revealed neither statistically significant main effects for bolus type nor interactions between gender and age. For between-subject effects, there was no main effect for gender, but there was a large main effect for age, and a gender and age interaction. In the group with COPD, there were no significant differences across bolus types; however, PO measures were consistently lower than the healthy groups for all bolus types. Healthy adults exhibited stable PO levels across bolus types. Adults with COPD, although exhibiting significantly lower PO levels, also remained stable. For clinicians who monitor PO measures, these results offer a more comprehensive understanding of the nature of these measures during swallowing in these groups.

Analysis of wearable time series data in endocrine and metabolic research.

Many hormones in the body oscillate with different frequencies and amplitudes, creating a dynamic environment that is essential to maintain health. In humans, disruptions to these rhythms are strongly associated with increased morbidity and mortality. While mathematical models can help us understand rhythm misalignment, translating this insight into personalised healthcare technologies requires solving additional challenges. Here, we discuss how combining minimally invasive, high-frequency biosampling technologies with wearable devices can assist the development of hormonal surrogates. We review bespoke algorithms that can help analyse multidimensional, noisy, time series data and identify wearable signals that could constitute clinical proxies of endocrine rhythms. These techniques can support the development of computational biomarkers to support the diagnosis and management of endocrine and metabolic conditions.

Localized Induction of Gene Expression in Embryonic Stem Cell Aggregates Using Holographic Optical Tweezers to Create Biochemical Gradients.

Three-dimensional (3D) cell models that mimic the structure and function of native tissues are enabling more detailed study of physiological and pathological mechanisms in vitro. We have previously demonstrated the ability to build and manipulate 3D multicellular microscopic structures using holographic optical tweezers (HOTs). Here, we show the construction of a precisely patterned 3D microenvironment and biochemical gradient model consisting of mouse embryoid bodies (mEBs) and polymer microparticles loaded with retinoic acid (RA), embedded in a hydrogel. We demonstrate discrete, zonal expression of the RA-inducible protein Stra8 within mEBs in response to release of RA from polymer microparticles, corresponding directly to the defined 3D positioning of the microparticles using HOTs. These results demonstrate the ability of this technology to create chemical microgradients at definable length scales and to elicit, with fidelity and precision, specific biological responses. This technique can be used in the study of in vitro microenvironments to enable new insights on 3D cell models, their cellular assembly, and the delivery of drug or biochemical molecules for engineering and interrogation of functional and morphogenic responses. Graphical abstract.

Completing the ß,γ-CXY-dNTP Stereochemical Probe Toolkit: Synthetic Access to the dCTP Diastereomers and 31P and 19F NMR Correlations with Absolute Configurations.

Nucleoside 5'-triphosphate (dNTP) analogues in which the ß,γ-oxygen is mimicked by a CXY group (ß,γ-CXY-dNTPs) have provided information about DNA polymerase catalysis and fidelity. Definition of CXY stereochemistry is important to elucidate precise binding modes. We previously reported the (R)- and (S)-ß,γ-CHX-dGTP diastereomers (X = F, Cl), prepared via P,C-dimorpholinamide CHCl (6a, 6b) and CHF (7a, 7b) bisphosphonates (BPs) equipped with an (R)-mandelic acid as a chiral auxiliary, with final deprotection using H2/Pd. This method also affords the ß,γ-CHCl-dTTP (11a, 11b), ß,γ-CHF (12a, 12b), and ß,γ-CHCl (13a, 13b) dATP diastereomers as documented here, but the reductive deprotection step is not compatible with dCTP or the bromo substituent in ß,γ-CHBr-dNTP analogues. To complete assembly of the toolkit, we describe an alternative synthetic strategy featuring ethylbenzylamine or phenylglycine-derived chiral BP synthons incorporating a photolabile protecting group. After acid-catalyzed removal of the (R)-(+)-α-ethylbenzylamine auxiliary, coupling with activated dCMP and photochemical deprotection, the individual diastereomers of ß,γ-CHBr- (33a, 33b), ß,γ-CHCl- (34a, 34b), ß,γ-CHF-dCTP (35a, 35b) were obtained. The ß,γ-CH(CH3)-dATPs (44a, 44b) were obtained using a methyl (R)-(-)-phenylglycinate auxiliary. 31P and 19F NMR Δδ values are correlated with CXY stereochemistry and pKa2-4 values for 13 CXY-bisphosphonic acids and imidodiphosphonic acid are tabulated.

Corticosteroids in Pediatric Heart Surgery: Myth or Reality.

Background: Corticosteroids have been administered prophylactically for more than 60 years in pediatric heart surgery, however, their use remains a matter of debate. There are three main indications for corticosteroid use in pediatric heart surgery with the use of cardiopulmonary bypass (CPB): (1) to blunt the systemic inflammatory response (SIRS) induced by the extracorporeal circuit; (2) to provide perioperative supplementation for presumed relative adrenal insufficiency; (3) for the presumed neuroprotective effect during deep hypothermic circulatory arrest operations. This review discusses the current evidence behind the use of corticosteroids in these three overlapping areas. Materials and Methods: We conducted a structured research of the literature using PubMed and MEDLINE databases to November 2017 and additional articles were identified by cross-referencing. Results: The evidence suggests that there is no correlation between the effect of corticosteroids on inflammation and their effect on clinical outcome. Due to the limitations of the available evidence, it remains unclear if corticosteroids have an impact on early post-operative outcomes or if there are any long-term effects. There is a limited understanding of the hypothalamic-pituitary-adrenal axis function during cardiac surgery in children. The neuroprotective effect of corticosteroids during deep hypothermic circulatory arrest surgery is controversial. Conclusions: The utility of steroid administration for pediatric heart surgery with the use of CPB remains a matter of debate. The effect on early and late outcomes requires clarification with a large multicenter randomized trial. More research into the understanding of the adrenal response to surgery in children and the effect of corticosteroids on brain injury is warranted.

Measures of behavioral function predict duration of video game play: Utilization of the Video Game Functional Assessment - Revised.

Background Internet gaming disorder (IGD) was introduced in the DSM-5 as a way of identifying and diagnosing problematic video game play. However, the use of the diagnosis is constrained, as it shares criteria with other addictive orders (e.g., pathological gambling). Aims Further work is required to better understand IGD. One potential avenue of investigation is IGD's relationship to the primary reinforcing behavioral functions. This study explores the relationship between duration of video game play and the reinforcing behavioral functions that may motivate or maintain video gaming. Methods A total of 499 video game players began the online survey, with complete data from 453 participants (85% white and 28% female), were analyzed. Individuals were placed into five groups based on self-reported hours of video gaming per week, and completed the Video Game Functional Assessment - Revised (VGFA-R). Results The results demonstrated the escape and social attention function were significant in predicting duration of video game play, whereas sensory and tangible were not significant. Conclusion Future implications of the VGFA-R and behaviorally based research are discussed.

Precision assembly of complex cellular microenvironments using holographic optical tweezers.

The accurate study of cellular microenvironments is limited by the lack of technologies that can manipulate cells in 3D at a sufficiently small length scale. The ability to build and manipulate multicellular microscopic structures will facilitate a more detailed understanding of cellular function in fields such as developmental and stem cell biology. We present a holographic optical tweezers based technology to accurately generate bespoke cellular micro-architectures. Using embryonic stem cells, 3D structures of varying geometries were created and stabilized using hydrogels and cell-cell adhesion methods. Control of chemical microenvironments was achieved by the temporal release of specific factors from polymer microparticles positioned within these constructs. Complex co-culture micro-environmental analogues were also generated to reproduce structures found within adult stem cell niches. The application of holographic optical tweezers-based micromanipulation will enable novel insights into biological microenvironments by allowing researchers to form complex architectures with sub-micron precision of cells, matrices and molecules.

Single-stranded DNA scanning and deamination by APOBEC3G cytidine deaminase at single molecule resolution.

APOBEC3G (Apo3G) is a single-stranded (ss)DNA cytosine deaminase that eliminates HIV-1 infectivity by converting C → U in numerous small target motifs on the minus viral cDNA. Apo3G deaminates linear ssDNA in vitro with pronounced spatial asymmetry favoring the 3' → 5' direction. A similar polarity observed in vivo is believed responsible for initiating localized C → T mutational gradients that inactivate the virus. When compared with double-stranded (ds)DNA scanning enzymes, e.g. DNA glycosylases that excise rare aberrant bases, there is a paucity of mechanistic studies on ssDNA scanning enzymes. Here, we investigate ssDNA scanning and motif-targeting mechanisms for Apo3G using single molecule Förster resonance energy transfer. We address the specific issue of deamination asymmetry within the general context of ssDNA scanning mechanisms and show that Apo3G scanning trajectories, ssDNA contraction, and deamination efficiencies depend on motif sequence, location, and ionic strength. Notably, we observe the presence of bidirectional quasi-localized scanning of Apo3G occurring proximal to a 5' hot motif, a motif-dependent DNA contraction greatest for 5' hot > 3' hot > 5' cold motifs, and diminished mobility at low salt. We discuss the single molecule Förster resonance energy transfer data in terms of a model in which deamination polarity occurs as a consequence of Apo3G binding to ssDNA in two orientations, one that is catalytically favorable, with the other disfavorable.

α-Azido bisphosphonates: synthesis and nucleotide analogues.

The first examples of α-azido bisphosphonates [(RO)(2)P(O)](2)CXN(3) (1, R = i-Pr, X = Me; 2, R = i-Pr, X = H; 3, R = H, X = Me; 4, R = H, X = H) and corresponding ß,γ-CXN(3) dGTP (5-6) and α,ß-CXN(3) dATP (7-8) analogues are described. The individual diastereomers of 7 (7a/b) were obtained by HPLC separation of the dADP synthetic precursor (14a/b).

Synthesis and biological evaluation of fluorinated deoxynucleotide analogs based on bis-(difluoromethylene)triphosphoric acid.

It is difficult to overestimate the importance of nucleoside triphosphates in cellular chemistry: They are the building blocks for DNA and RNA and important sources of energy. Modifications of biologically important organic molecules with fluorine are of great interest to chemists and biologists because the size and electronegativity of the fluorine atom can be used to make defined structural alterations to biologically important molecules. Although the concept of nonhydrolyzable nucleotides has been around for some time, the progress in the area of modified triphosphates was limited by the lack of synthetic methods allowing to access bisCF(2)-substituted nucleotide analogs-one of the most interesting classes of nonhydrolyzable nucleotides. These compounds have "correct" polarity and the smallest possible steric perturbation compared to natural nucleotides. No other known nucleotides have these advantages, making bisCF(2)-substituted analogs unique. Herein, we report a concise route for the preparation of hitherto unknown highly acidic and polybasic bis(difluoromethylene)triphosphoric acid 1 using a phosphorous(III)/phosphorous(V) interconversion approach. The analog 1 compared to triphosphoric acid is enzymatically nonhydrolyzable due to substitution of two bridging oxygen atoms with CF(2) groups, maintaining minimal perturbations in steric bulkiness and overall polarity of the triphosphate polyanion. The fluorinated triphosphoric acid 1 was used for the preparation of the corresponding fluorinated deoxynucleotides (dNTPs). One of these dNTP analogs (dT) was demonstrated to fit into DNA polymerase beta (DNA pol beta) binding pocket by obtaining a 2.5 A resolution crystal structure of a ternary complex with the enzyme. Unexpected dominating effect of triphosphate/Mg(2+) interaction over Watson-Crick hydrogen bonding was found and discussed.

Halogenated beta,gamma-methylene- and ethylidene-dGTP-DNA ternary complexes with DNA polymerase beta: structural evidence for stereospecific binding of the fluoromethylene analogues.

Beta,gamma-fluoromethylene analogues of nucleotides are considered to be useful mimics of the natural substrates, but direct structural evidence defining their active site interactions has not been available, including the influence of the new chiral center introduced at the CHF carbon, as in beta,gamma-fluoromethylene-dGTP, which forms an active site complex with DNA polymerase beta, a repair enzyme that plays an important role in base excision repair (BER) and oncogenesis. We report X-ray crystallographic results for a series of beta,gamma-CXY dGTP analogues, where X,Y = H, F, Cl, Br, and/or CH(3). For all three R/S monofluorinated analogues examined (CHF, 3/4; CCH(3)F, 13/14; CClF 15/16), a single CXF-diastereomer (3, 13, 16) is observed in the active site complex, with the CXF fluorine atom at a approximately 3 A (bonding) distance to a guanidinium N of Arg183. In contrast, for the CHCl, CHBr, and CHCH(3) analogues, both diasteromers (6/7, 8/9, 10/11) populate the dGTP site in the enzyme complex about equally. The structures of the bound dichloro (5) and dimethyl (12) analogue complexes indicate little to no steric effect on the placement of the bound nucleotide backbone. The results suggest that introduction of a single fluorine atom at the beta,gamma-bridging carbon atom of these dNTP analogues enables a new, stereospecific interaction within the preorganized active site complex that is unique to fluorine. The results also provide the first diverse structural data set permitting an assessment of how closely this class of dNTP analogues mimics the conformation of the parent nucleotide within the active site complex.

Alpha,beta-difluoromethylene deoxynucleoside 5'-triphosphates: a convenient synthesis of useful probes for DNA polymerase beta structure and function.

Alpha,beta-difluoromethylene deoxynucleoside 5'-triphosphates (dNTPs, N = A or C) are advantageously obtained via phosphorylation of corresponding dNDP analogues using catalytic ATP, PEP, nucleoside diphosphate kinase, and pyruvate kinase. DNA pol beta K(d) values for the alpha,beta-CF(2) and unmodified dNTPs, alpha,beta-NH dUTP, and the alpha,beta-CH(2) analogues of dATP and dGTP are discussed in relation to the conformations of alpha,beta-CF(2) dTTP versus alpha,beta-NH dUTP bound into the enzyme active site.

DNA polymerase beta fidelity: halomethylene-modified leaving groups in pre-steady-state kinetic analysis reveal differences at the chemical transition state.

The mechanism of DNA polymerase beta-catalyzed nucleotidyl transfer consists of chemical steps involving primer 3' OH deprotonation, nucleophilic attack, and pyrophosphate leaving-group elimination, preceded by dNTP binding which induces a large-amplitude conformational change for Watson-Crick nascent base pairs. Ambiguity in the nature of the rate-limiting step and active-site structural differences between correct and incorrect base-paired transition states remain obstacles to understanding DNA replication fidelity. Analogues of dGTP where the beta-gamma bridging oxygen is replaced with fluorine-substituted methylene groups have been shown to probe the contribution of leaving-group elimination to the overall catalytic rate (Biochemistry 46, 461-471). Here, the analysis is expanded substantially to include a broad range of halogen substituents with disparate steric and electronic properties. Evaluation of linear free energy relationships for incorporation of dGTP analogues opposite either template base C or T reveals a strong correlation of log(kpol) to leaving group pKa. Significantly different kpol behavior is observed with a subset of the analogues, with magnitude dependent on the identity of the nascent base pair. This observation, and the absence of an analogous effect on ground state analogue binding (Kd values), points to active-site structural differences at the chemical transition state. Reduced catalysis with bulky halo-containing substrates is manifested in the fidelity of T-G incorporation, where the CCl2-bridging analogue shows a 27-fold increase in fidelity over the natural dGTP. Solvent pH and deuterium isotope-effect data are also used to evaluate mechanistic differences between correct and mispaired incorporation.