Urinary ATP and visualization of intracellular bacteria: a superior diagnostic marker for recurrent UTI in renal transplant recipients?

Renal transplant recipients (RTR) are highly susceptible to urinary tract infections (UTIs) with over 50% of patients having at least one UTI within the first year. Yet it is generally acknowledged that there is considerable insensitivity and inaccuracy in routine urinalysis when screening for UTIs. Thus a large number of transplant patients with genuine urine infections may go undiagnosed and develop chronic recalcitrant infections, which can be associated with graft loss and morbidity. Given a recent study demonstrating ATP is released by urothelial cells in response to bacteria exposure, possibly acting at metabotropic P2Y receptors mediating a proinflammatory response, we have investigated alternative, and possibly more appropriate, urinalysis techniques in a cohort of RTRs.Mid-stream urine (MSU) samples were collected from 53 outpatient RTRs. Conventional leukocyte esterase and nitrite dipstick tests, and microscopic pyuria counts (in 1 ?l), ATP concentration measurements, and identification of intracellular bacteria in shed urothelial cells, were performed on fresh unspun samples and compared to ‘gold-standard’ bacterial culture results.Of the 53 RTRs, 22% were deemed to have a UTI by ‘gold-standard’ conventional bacteria culture, whereas 87%, 8% and 4% showed evidence of UTIs according to leukocyte esterase dipstick, nitrite dipstick, and a combination of both dipsticks, respectively. Intracellular bacteria were visualized in shed urothelial cells of 44% of RTRs, however only 1 of the 23 RTRs (44%) was deemed to have a UTI by conventional bacteria culture. A significant association of the ‘gold-standard’ test with urinary ATP concentration combined with visualization of intracellular bacteria in shed urothelial cells was determined using the Fisher’s exact test.It is apparent that standard bedside tests for UTIs give variable results and that seemingly quiescent bacteria in urothelial cells are very common in RTRs and may represent a focus of subclinical infection. Furthermore, our results suggest urinary ATP concentration combined with detection of intracellular bacteria in shed urinary epithelial cells may be a sensitive means by which to detect ‘occult’ infection in RTRs

Inflammatory mediators act at renal pericytes to elicit contraction of vasa recta and reduce pericyte density along the kidney medullary vascular network

Introduction: Regardless of initiating cause, renal injury promotes a potent pro-inflammatory environment in the outer medulla and a concomitant sustained decrease in medullary blood flow (MBF). This decline in MBF is believed to be one of the critical events in the pathogenesis of acute kidney injury (AKI), yet the precise cellular mechanism underlying this are still to be fully elucidated. MBF is regulated by contractile pericyte cells that reside on the descending vasa recta (DVR) capillaries, which are the primary source of blood flow to the medulla. Methods: Using the rat and murine live kidney slice models, we investigated the acute effects of key medullary inflammatory mediators TNF-α, IL-1β, IL-33, IL-18, C3a and C5a on vasa recta pericytes, the effect of AT1-R blocker Losartan on pro-inflammatory mediator activity at vasa recta pericytes, and the effect of 4-hour sustained exposure on immunolabelled NG2+ pericytes. Results and discussion: Exposure of rat and mouse kidney slices to TNF-α, IL-18, IL-33, and C5a demonstrated a real-time pericyte-mediated constriction of DVR. When pro-inflammatory mediators were applied in the presence of Losartan the inflammatory mediator-mediated constriction that had previously been observed was significantly attenuated. When live kidney slices were exposed to inflammatory mediators for 4-h, we noted a significant reduction in the number of NG2+ positive pericytes along vasa recta capillaries in both rat and murine kidney slices. Data collected in this study demonstrate that inflammatory mediators can dysregulate pericytes to constrict DVR diameter and reduce the density of pericytes along vasa recta vessels, further diminishing the regulatory capacity of the capillary network. We postulate that preliminary findings here suggest pericytes play a role in AKI

Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ? 98 %). All samples were analyzed following injection (100 ?l) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 ?m, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2-30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min(-1) flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients

Inhibition of native 5-HT3 receptor-evoked contractions in Guinea pig and mouse ileum by antimalarial drugs

Quinine, Chloroquine and mefloquine are commonly used to treat malaria; however with associated gastrointestinal (GI) side-effects. These drugs act as antagonists at recombinant 5-HT3 receptors and modulate gut peristalsis. These gastrointestinal side effects may be the result of antagonism at intestinal 5-HT3 receptors. Ileum from male C57BL/6 mice and guinea pigs was mounted longitudinally in organ baths. Concentration-response curves for 5-HT and the selective 5-HT3 agonist 2-Me-5-HT were obtained with 5-HT (pEC50=7.57±0.33, 12) more potent (P=0.004) than 2-Me-5-HT (pEC50=5.45±0.58, n=5) in mouse ileum. There was no difference in potency of 5-HT (pEC50=5.42±0.15, n=8) and 2-Me-5-HT (pIC50=5.01±0.55, n=11) in guinea pig ileum (P>0.05). Quinine, Chloroquine or mefloquine was applied for 10 min and inhibitions prior to submaximal agonist application. In mouse ileum, quinine, chloroquine and mefloquine antagonised 5-HT-induced contractions (pIC50=4.9±0.17, n=7; 4.76±0.14,n=5; 6.21±0.2, n=4, respectively) with mefloquine most potent (P<0.05). Quinine, chloroquine and mefloquine antagonised 2-me-5-HT-induced contractions (pIC50=6.35±0.11,n=8; 4.64±0.2, n=7; 5.11± 0.22, n=6, respectively) with quinine most potent (P<0.05). In guinea-pig ileum, quinine, chloroquine and mefloquine antagonised 5-HT-induced contractions (pIC50=5.02±0.15, n=6; 4.54±0.1, n=7; 5.32±0.13, n=5, respectively) and 2-me-5-HT-induced contractions (pIC50=4.62±0.25, n=5; 4.56±0.14, n=6; 5.67±0.12, n=4, respectively) with chloroquine least potent against 5-HT and mefloquine most potent against 2-me-5-HT (P<0.05). These results support previous studies identifying anti-malarial drugs as antagonists at recombinant 5-HT3 receptors and may also demonstrate the ability of these drugs to influence native 5-HT3 receptor-evoked contractile responses which may account for their associated GI side-effects

Modeling Signal Propagation Mechanisms and Ligand-Based Conformational Dynamics of the Hsp90 Molecular Chaperone Full-Length Dimer

Hsp90 is a molecular chaperone essential for protein folding and activation in normal homeostasis and stress response. ATP binding and hydrolysis facilitate Hsp90 conformational changes required for client activation. Hsp90 plays an important role in disease states, particularly in cancer, where chaperoning of the mutated and overexpressed oncoproteins is important for function. Recent studies have illuminated mechanisms related to the chaperone function. However, an atomic resolution view of Hsp90 conformational dynamics, determined by the presence of different binding partners, is critical to define communication pathways between remote residues in different domains intimately affecting the chaperone cycle. Here, we present a computational analysis of signal propagation and long-range communication pathways in Hsp90. We carried out molecular dynamics simulations of the full-length Hsp90 dimer, combined with essential dynamics, correlation analysis, and a signal propagation model. All-atom MD simulations with timescales of 70 ns have been performed for complexes with the natural substrates ATP and ADP and for the unliganded dimer. We elucidate the mechanisms of signal propagation and determine “hot spots” involved in interdomain communication pathways from the nucleotide-binding site to the C-terminal domain interface. A comprehensive computational analysis of the Hsp90 communication pathways and dynamics at atomic resolution has revealed the role of the nucleotide in effecting conformational changes, elucidating the mechanisms of signal propagation. Functionally important residues and secondary structure elements emerge as effective mediators of communication between the nucleotide-binding site and the C-terminal interface. Furthermore, we show that specific interdomain signal propagation pathways may be activated as a function of the ligand. Our results support a “conformational selection model” of the Hsp90 mechanism, whereby the protein may exist in a dynamic equilibrium between different conformational states available on the energy landscape and binding of a specific partner can bias the equilibrium toward functionally relevant complexes

An intervention to support adherence to inhaled medication in adults with cystic fibrosis : the ACtiF research programme including RCT

Background People with cystic fibrosis frequently have low levels of adherence to inhaled medications. Objectives The objectives were to develop and evaluate an intervention for adults with cystic fibrosis to improve adherence to their inhaled medication. Design We used agile software methods to develop an online platform. We used mixed methods to develop a behaviour change intervention for delivery by an interventionist. These were integrated to become the CFHealthHub intervention. We undertook a feasibility study consisting of a pilot randomised controlled trial and process evaluation in two cystic fibrosis centres. We evaluated the intervention using an open-label, parallel-group randomised controlled trial with usual care as the control. Participants were randomised in a 1 : 1 ratio to intervention or usual care. Usual care consisted of clinic visits every 3 months. We undertook a process evaluation alongside the randomised controlled trial, including a fidelity study, a qualitative interview study and a mediation analysis. We undertook a health economic analysis using both a within-trial and model-based analysis. Setting The randomised controlled trial took place in 19 UK cystic fibrosis centres. Participants Participants were people aged ≥ 16 years with cystic fibrosis, on the cystic fibrosis registry, not post lung transplant or on the active transplant list, who were able to consent and not using dry-powder inhalers. Intervention People with cystic fibrosis used a nebuliser with electronic monitoring capabilities. This transferred data automatically to a digital platform. People with cystic fibrosis and clinicians could monitor adherence using these data, including through a mobile application (app). CFHealthHub displayed graphs of adherence data as well as educational and problem-solving information. A trained interventionist helped people with cystic fibrosis to address their adherence. Main outcome measures Randomised controlled trial – adjusted incidence rate ratio of pulmonary exacerbations meeting the modified Fuchs criteria over a 12-month follow-up period (primary outcome); change in percentage adherence; and per cent predicted forced expiratory volume in 1 second (key secondary outcomes). Process evaluation – percentage fidelity to intervention delivery, and participant and interventionist perceptions of the intervention. Economic modelling – incremental cost per quality-adjusted life-year gained. Results Randomised controlled trial – 608 participants were randomised to the intervention (n = 305) or usual care (n = 303). To our knowledge, this was the largest randomised controlled trial in cystic fibrosis undertaken in the UK. The adjusted rate of exacerbations per year (primary outcome) was 1.63 in the intervention and 1.77 in the usual-care arm (incidence rate ratio 0.96, 95% confidence interval 0.83 to 1.12; p = 0.638) after adjustment for covariates. The adjusted difference in mean weekly normative adherence was 9.5% (95% confidence interval 8.6% to 10.4%) across 1 year, favouring the intervention. Adjusted mean difference in forced expiratory volume in 1 second (per cent) predicted at 12 months was 1.4% (95% confidence interval –0.2% to 3.0%). No adverse events were related to the intervention. Process evaluation – fidelity of intervention delivery was high, the intervention was acceptable to people with cystic fibrosis, participants engaged with the intervention [287/305 (94%) attended the first intervention visit], expected mechanisms of action were identified and contextual factors varied between randomised controlled trial sites. Qualitative interviews with 22 people with cystic fibrosis and 26 interventionists identified that people with cystic fibrosis welcomed the objective adherence data as proof of actions to self and others, and valued the relationship that they built with the interventionists. Economic modelling – the within-trial analysis suggests that the intervention generated 0.01 additional quality-adjusted life-years at an additional cost of £865.91 per patient, leading to an incremental cost-effectiveness ratio of £71,136 per quality-adjusted life-year gained. This should be interpreted with caution owing to the short time horizon. The health economic model suggests that the intervention is expected to generate 0.17 additional quality-adjusted life-years and cost savings of £1790 over a lifetime (70-year) horizon; hence, the intervention is expected to dominate usual care. Assuming a willingness-to-pay threshold of £20,000 per quality-adjusted life-year gained, the probability that the intervention generates more net benefit than usual care is 0.89. The model results are dependent on assumptions regarding the duration over which costs and effects of the intervention apply, the impact of the intervention on forced expiratory volume in 1 second (per cent) predicted and the relationship between increased adherence and drug-prescribing levels. Limitations Number of exacerbations is a sensitive and valid measure of clinical change used in many trials. However, data collection of this outcome in this context was challenging and could have been subject to bias. It was not possible to measure baseline adherence accurately. It was not possible to quantify the impact of the intervention on the number of packs of medicines prescribed. Conclusions We developed a feasible and acceptable intervention that was delivered to fidelity in the randomised controlled trial. We observed no statistically significant difference in the primary outcome of exacerbation rates over 12 months. We observed an increase in normative adherence levels in a disease where adherence levels are low. The magnitude of the increase in adherence may not have been large enough to affect exacerbations. Future work Given the non-significant difference in the primary outcome, further research is required to explore why an increase in objective normative adherence did not reduce exacerbations and to develop interventions that reduce exacerbations. Trial registration Work package 3.1: Current Controlled Trials ISRCTN13076797. Work packages 3.2 and 3.3: Current Controlled Trials ISRCTN55504164

Assessing childhood maltreatment and mental health correlates of disordered eating profiles in a nationally representative sample of English females

PURPOSE: Previous research suggests that childhood maltreatment is associated with the onset of eating disorders (ED). In turn, EDs are associated with alternative psychopathologies such as depression and posttraumatic stress disorder (PTSD), and with suicidality. Moreover, it has been reported that various ED profiles may exist. The aim of the current study was to examine the profiles of disordered eating and the associations of these with childhood maltreatment and with mental health psychopathology. METHODS: The current study utilised a representative sample of English females (N = 4206) and assessed for the presence of disordered eating profiles using Latent Class Analysis. Multinomial logistic regression was implemented to examine the associations of childhood sexual and physical abuse with the disordered eating profiles and the associations of these with PTSD, depression and suicidality. RESULTS: Results supported those of previous findings in that we found five latent classes of which three were regarded as disordered eating classes. Significant relationships were found between these and measures of childhood trauma and mental health outcomes. CONCLUSIONS: Childhood sexual and physical abuse increased the likelihood of membership in disordered eating classes and these in turn increased the likelihood of adverse mental health and suicidal outcomes

Multi-ancestry genome-wide association study of major depression aids locus discovery, fine mapping, gene prioritization and causal inference.

Most genome-wide association studies (GWAS) of major depression (MD) have been conducted in samples of European ancestry. Here we report a multi-ancestry GWAS of MD, adding data from 21 cohorts with 88,316 MD cases and 902,757 controls to previously reported data. This analysis used a range of measures to define MD and included samples of African (36% of effective sample size), East Asian (26%) and South Asian (6%) ancestry and Hispanic/Latin American participants (32%). The multi-ancestry GWAS identified 53 significantly associated novel loci. For loci from GWAS in European ancestry samples, fewer than expected were transferable to other ancestry groups. Fine mapping benefited from additional sample diversity. A transcriptome-wide association study identified 205 significantly associated novel genes. These findings suggest that, for MD, increasing ancestral and global diversity in genetic studies may be particularly important to ensure discovery of core genes and inform about transferability of findings

Climatic and topographic changes since the Miocene influenced the diversification and biogeography of the tent tortoise (Psammobates tentorius) species complex in Southern Africa

Background: Climatic and topographic changes function as key drivers in shaping genetic structure and cladogenic radiation in many organisms. Southern Africa has an exceptionally diverse tortoise fauna, harbouring one-third of the world’s tortoise genera. The distribution of Psammobates tentorius (Kuhl, 1820) covers two of the 25 biodiversity hotspots in the world, the Succulent Karoo and Cape Floristic Region. The highly diverged P. tentorius represents an excellent model species for exploring biogeographic and radiation patterns of reptiles in Southern Africa. Results: We investigated genetic structure and radiation patterns against temporal and spatial dimensions since the Miocene in the Psammobates tentorius species complex, using multiple types of DNA markers and niche modelling analyses. Cladogenesis in P. tentorius started in the late Miocene (11.63–5.33 Ma) when populations dispersed from north to south to form two geographically isolated groups. The northern group diverged into a clade north of the Orange River (OR), followed by the splitting of the group south of the OR into a western and an interior clade. The latter divergence corresponded to the intensifcation of the cold Benguela current, which caused western aridifcation and rainfall seasonality. In the south, tectonic uplift and subsequent exhumation, together with climatic fuctuations seemed responsible for radiations among the four southern clades since the late Miocene. We found that each clade occurred in a habitat shaped by diferent climatic parameters, and that the niches difered substantially among the clades of the northern group but were similar among clades of the southern group. Conclusion: Climatic shifts, and biome and geographic changes were possibly the three major driving forces shaping cladogenesis and genetic structure in Southern African tortoise species. Our results revealed that the cladogenesis of the P. tentorius species complex was probably shaped by environmental cooling, biome shifts and topographic uplift in Southern Africa since the late Miocene. The Last Glacial Maximum (LGM) may have impacted the distribution of P. tentorius substantially. We found the taxonomic diversify of the P. tentorius species complex to be highest in the Greater Cape Floristic Region. All seven clades discovered warrant conservation attention, particularly Ptt-B–Ptr, Ptt-A and Pv-

Understanding biomolecular motion, recognition, and allostery by use of conformational ensembles

We review the role conformational ensembles can play in the analysis of biomolecular dynamics, molecular recognition, and allostery. We introduce currently available methods for generating ensembles of biomolecules and illustrate their application with relevant examples from the literature. We show how, for binding, conformational ensembles provide a way of distinguishing the competing models of induced fit and conformational selection. For allostery we review the classic models and show how conformational ensembles can play a role in unravelling the intricate pathways of communication that enable allostery to occur. Finally, we discuss the limitations of conformational ensembles and highlight some potential applications for the future