Traditional small molecule drug discovery is a time consuming and costly endeavor. High-throughput chemical screening can only assess a tiny fraction of drug-like chemical space. The strong predictive power of modern machine learning methods for virtual chemical screening enables training models on known active and inactive compounds and extrapolating to much larger chemical libraries. However, there has been limited experimental validation of these models' extensibility toward practical applications on large commercially-available or synthesize-on-demand chemical libraries. Through a prospective evaluation with the bacterial protein-protein interaction PriA-SSB, we demonstrate that ligand-based virtual screening can identify many chemically-diverse active compounds in a large commercial library. We use cross validation to compare many supervised learning models and select a random forest classifier as the best model for this target. When predicting the activity of more than 8 million compounds from Aldrich Market Select, the random forest substantially outperforms a naïve baseline based on chemical structure similarity. 48% of the random forest's 701 selected compounds are active. The random forest model easily scales to score one billion compounds from the synthesize-on-demand Enamine REAL database. We tested 68 chemically-diverse top predictions from Enamine REAL and observed 31 hits (46%), including one with an IC50 value of 1.3 µM.

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.

Functional data here offer novel insight into the vasoactive activity of the renal GABA/glutamate system. These data show that activation of endogenous GABA and glutamate receptors in the kidney significantly alters microvessel diameter. Furthermore, the results show that these antiepileptic drugs are as potentially challenging to the kidney as nonsteroidal anti-inflammatory drugs.

Fragile X syndrome (FXS) is the most common inherited cause of autism and intellectual disability. The majority of FXS cases are caused by transcriptional repression of the FMR1 gene due to epigenetic changes that are not recapitulated in current animal disease models. FXS patient induced pluripotent stem cell (iPSC)-derived gene edited reporter cell lines enable novel strategies to discover reactivators of FMR1 expression in human cells on a much larger scale than previously possible. Here, we describe the workflow using FXS iPSC-derived neural cell lines to conduct a massive, unbiased screen for small molecule activators of the FMR1 gene. The proof-of-principle methodology demonstrates the utility of human stem-cell-based methodology for the untargeted discovery of reactivators of the human FMR1 gene that can be applied to other diseases.

Regulation of microvascular blood flow in the bladder has historically been associated with regulation by pre‐capillary arterioles and more recently to post‐capillary venules. Although pericytes are known to regulate capillary vessel diameter in various capillary beds (e.g. brain, retina, heart and kidney), their role in regulating bladder capillary diameter remains controversial.We have developed a mouse full‐thickness live bladder tissue model in which the microvasculature of the mucosa can be visualised in situ, enabling the regulation of suburothelial capillary diameter to be investigated. Bladder tissue viability was first investigated by fluorescence imaging of tissue labelled with Hoechst and propidium iodide to determine the live to dead cell ratio. Data indicated that tissue was viable in situ for up to 5 hours (>60%; P>0.05; n = 3 per time point). Real time DIC images of live tissue sections were collected for all experiments in which live tissue was exposed to vasoactive agents to investigate the pericyte‐mediated regulation of capillary diameter. Images were analysed off line and vessel diameter was measured at both pericyte and non‐pericyte sites along a capillary for each experiment. Angiotensin II (100 nM) and endothelin I (1 nM) evoked a pericyte‐mediated vasoconstriction of suburothelial capillaries at pericyte sites (13.3 ± 5.8%, 22.0 ± 9.7%) that was significantly greater (p<0.05, n = 6 per drug) than that measured at non‐pericyte sites (3.2 ± 2.7%, 6.0 ± 3.4%). Prostaglandin E2 (30 μM) evoked a pericyte‐mediated dilation of capillaries (12.1 ± 2.6%) that was significantly greater than that measured at non‐pericyte sites (2.3 ± 2.3%, p = 0.03, n = 3).In this study we therefore demonstrate that suburothelial capillary pericytes act to regulate capillary diameter in response to agents previously reported to regulate capillary beds of other organs. This indicates that in addition to smooth muscle‐mediated regulation of upstream vessels in the bladder, spatial regulation of capillary diameter downstream of these vessels also occurs in the bladder.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Antimicrobial resistance is a global health crisis and few novel antimicrobials have been discovered in recent decades. Natural products, particularly from Streptomyces, are the source of most antimicrobials, yet discovery campaigns focusing on Streptomyces from the soil largely rediscover known compounds. Investigation of understudied and symbiotic sources has seen some success, yet no studies have systematically explored microbiomes for antimicrobials. Here we assess the distinct evolutionary lineages of Streptomyces from insect microbiomes as a source of new antimicrobials through large-scale isolations, bioactivity assays, genomics, metabolomics, and in vivo infection models. Insect-associated Streptomyces inhibit antimicrobial-resistant pathogens more than soil Streptomyces. Genomics and metabolomics reveal their diverse biosynthetic capabilities. Further, we describe cyphomycin, a new molecule active against multidrug resistant fungal pathogens. The evolutionary trajectories of Streptomyces from the insect microbiome influence their biosynthetic potential and ability to inhibit resistant pathogens, supporting the promise of this source in augmenting future antimicrobial discovery.

The newly recognized sensory role of bladder urothelium has generated intense interest in identifying its novel sensory molecules. Sensory receptor TRPV4 may serve such function. However, specific and physiologically relevant tissue actions of TRPV4, stretch-independent responses, and underlying mechanisms are unknown and its role in human conditions has not been examined. Here we showed TRPV4 expression in guinea-pig urothelium, suburothelium, and bladder smooth muscle, with urothelial predominance. Selective TRPV4 activation without stretch evoked significant ATP release-key urothelial sensory process, from live mucosa tissue, full-thickness bladder but not smooth muscle, and sustained muscle contractions. ATP release was mediated by Ca2+-dependent, pannexin/connexin-conductive pathway involving protein tyrosine kinase, but independent from vesicular transport and chloride channels. TRPV4 activation generated greater Ca2+ rise than purinergic activation in urothelial cells. There was intrinsic TRPV4 activity without exogeneous stimulus, causing ATP release. TRPV4 contributed to 50% stretch-induced ATP release. TRPV4 activation also triggered superoxide release. TRPV4 expression was increased with aging. Human bladder mucosa presented similarities to guinea pigs. Overactive bladders exhibited greater TRPV4-induced ATP release with age dependence. These data provide the first evidence in humans for the key functional role of TRPV4 in urothelium with specific mechanisms and identify TRPV4 up-regulation in aging and overactive bladders.

In the kidney, purinergic (P2) receptor-mediated ATP signaling has been shown to be an important local regulator of epithelial sodium transport. Appropriate sodium regulation is crucial for blood pressure (BP) control and disturbances in sodium balance can lead to hypo- or hypertension. Links have already been established between P2 receptor signaling and the development of hypertension, attributed mainly to vascular and/or inflammatory effects. A transgenic mouse model with deletion of the P2X4 receptor (P2X4-/- ) is known to have hypertension, which is thought to reflect endothelial dysfunction and impaired nitric oxide (NO) release. However, renal function in this model has not been characterized; moreover, studies in vitro have shown that the P2X4 receptor can regulate renal epithelial Na+ channel (ENaC) activity. Therefore, in the present study we investigated renal function and sodium handling in P2X4-/- mice, focusing on ENaC-mediated Na+ reabsorption. We confirmed an elevated BP in P2X4-/- mice compared with wild-type mice, but found that ENaC-mediated Na+ reabsorption is no different from wild-type and does not contribute to the raised BP observed in the knockout. However, when P2X4-/- mice were placed on a low sodium diet, BP normalized. Plasma aldosterone concentration tended to increase according to sodium restriction status in both genotypes; in contrast to wild-types, P2X4-/- mice did not show an increase in functional ENaC activity. Thus, although the increased BP in P2X4-/- mice has been attributed to endothelial dysfunction and impaired NO release, there is also a sodium-sensitive component.

Virtual (computational) high-throughput screening provides a strategy for prioritizing compounds for experimental screens, but the choice of virtual screening algorithm depends on the data set and evaluation strategy. We consider a wide range of ligand-based machine learning and docking-based approaches for virtual screening on two protein-protein interactions, PriA-SSB and RMI-FANCM, and present a strategy for choosing which algorithm is best for prospective compound prioritization. Our workflow identifies a random forest as the best algorithm for these targets over more sophisticated neural network-based models. The top 250 predictions from our selected random forest recover 37 of the 54 active compounds from a library of 22,434 new molecules assayed on PriA-SSB. We show that virtual screening methods that perform well on public data sets and synthetic benchmarks, like multi-task neural networks, may not always translate to prospective screening performance on a specific assay of interest.

In structure-based virtual screening, compound ranking through a consensus of scores from a variety of docking programs or scoring functions, rather than ranking by scores from a single program, provides better predictive performance and reduces target performance variability. Here we compare traditional consensus scoring methods with a novel, unsupervised gradient boosting approach. We also observed increased score variation among active ligands and developed a statistical mixture model consensus score based on combining score means and variances. To evaluate performance, we used the common performance metrics ROCAUC and EF1 on 21 benchmark targets from DUD-E. Traditional consensus methods, such as taking the mean of quantile normalized docking scores, outperformed individual docking methods and are more robust to target variation. The mixture model and gradient boosting provided further improvements over the traditional consensus methods. These methods are readily applicable to new targets in academic research and overcome the potentially poor performance of using a single docking method on a new target.

Objective To characterise the microbiome in healthy women with no bladder symptoms and to compare this to the bladder microbiome in patients with overactive bladder syndrome (OAB). Study design MSU specimens from 63 women with OAB were compared to urine from 35 controls. Urine was centrifuged and the resulting sediment pellet was re-suspended in supernatant and plated under aerobic conditions for 48 h and anaerobic conditions for 7 days. Each morphologically distinct colony was purity plated. Bacterial colonies were lysed and polymerase chain reaction undertaken to amplify the 16 s ribosomal RNA gene. This DNA was purified and sequenced allowing identification of bacterial genera. Results The mean number of different bacterial genera was 5.0 in both controls and OAB patients (p = 0.99). The uropathogenic bacteria Proteus (P = 0.01) was more commonly isolated from women with OAB. The genus lactobacillus was present less commonly in urine from OAB patients when compared to urine taken from controls (p = 0.02). Overall the most commonly grown bacteria were staphylococcus (grown in 59% of samples), streptococccus (51%), corynebacterium (37%) and lactobacillus (28%). A total of 95 different genera were identified from the urine samples. Conclusion The female human bladder has a diverse microbiome with stastistically significant differences between bacterial species present in OAB patients and controls.

If you believe this document infringes copyright then please contact the KAR admin team with the take-down information provided at http://kar.kent.ac.uk/contact.html Citation for published version Contreras-Sanz, Alberto and Krska, Louise and Balachandran, Aswini A. and Curtiss, Natasha L. and Khasriya, Rajvinder and Kelley, Stephen P. and Strutt, Matthew and Gill, Hardyal S. and Taylor, Kevin and Mansfield, Kylie J. and Wu, Changhao and Peppiatt-Wildman, Claire M. and Malone-Lee, James and Duckett, Jonathan and Wildman, Scott S.P. (2016) Altered urothelial

The innate immune system is thought to play a role in the pathogenesis of kidney diseases, such as acute kidney injury (AKI), and yet the precise mechanisms involved are unknown [1]. Mitochondrial membrane potential (ΔΨm), reactive oxygen species (ROS) and necrosis are thought to be involved in the injury process of AKI [2] and dysregulation of pericyte function has been associated with ischemic injury, AKI and progression to chronic kidney disease [3]. Here we use a rat live kidney slice model [4] in combination with multiphoton microscopy, to investigate whether various components of the innate immune system alter ΔΨm and/or induce production of mitochondrial ROS and cell death in the renal medulla, to assess their effect on cell function. Kidney slices were loaded with the appropriate fluorescent dye; tetramethylrhodamine methyl ester (TMRM), mitoSOX and propidium iodide (PI), respectively. Tissue was then superfused with complement component 5a (C5a), interleukin‐33 (IL‐33) and tumour necrosis factor alpha (TNF‐α), and changes in fluorescent signal were recorded via imaging techniques. C5a evoked a significant increase (34.73% ± 4.56%, 40.87% ± 8.34%, 34.40% ± 7.12%) in TMRM signal in endothelial (E), pericyte (P) and tubule cells (T), respectively, indicating membrane hyperpolarisation. Similar increases in TMRM signal were measured in response to IL‐33 (E: 26.25% ± 2.68%, P: 29.27% ± 8.82, T: 20.79% ± 2.61%) and TNF‐α (E: 44.72% ± 3.72%, P: 52.64% ± 4.29%, T: 54.75% ± 6.14%). C5a also evoked an increase (24.96% ± 5.87%, 31.41% ± 7.70%, 50.14% ± 7.69%) in mitoSOX signal in E, P and T cells, respectively, indicating an increase in mitochondrial ROS, associated with membrane hyperpolarization. Both C5a and TNF‐α evoked increases in mitoSOX fluorescence (E: 44.59% ± 8.59%, P: 30.92% ± 9.07%, T: 26.25% ± 3.37% and E: 61.10% ± 9.72%, P: 51.51% ± 12.08%, T: 70.41% ± 9.25% respectively). Furthermore, C5a also evoked a significant increase (22.74% ± 4.80%) in propidium iodide fluorescent cells across the tissue slice, as did C5a (15.58% ± 3.81%) and TNF‐α (17.02% ± 4.78%) when compared to PSS control experiments (1.58% ± 0.35%), indicating an increase in necrotic cells. We conclude that the innate immune system components C5a, IL‐33 and TNF‐α act at medullary endothelial, pericyte and tubule cells causing membrane hyperpolarisation, and increases in mitochondrial ROS resulting in an increase in necrotic cells. These initial observations provide preliminary evidence that suggests C5a, IL‐33 and TNF‐α may mediate some of the pathophysiological changes thought to underlie AKI. Support or Funding Information Kidney Research UK

Overactive bladder (OAB) is a common syndrome characterized by increased frequency, urinary urgency, urinary incontinence and nocturia. The cause of idiopathic OAB is unknown, however, a recent investigation has shown that antibiotics may reduce the symptoms of OAB in some patients (1). There is also an increasing body of evidence to suggest that the human bladder has a microbiome, missed by conventional urinalysis and culture (2). Consequently, infection may be a possible cause for the symptoms of OAB in some patients affected by this syndrome.We hypothesise that changes in the urinary tract microbiome are responsible for the symptoms of OAB. The aim of this investigation is to compare the species of bacteria in the urine of patients with OAB and age‐matched controls.Urine samples (5 ml), taken from females aged 35–87 via MSU sampling techniques, were centrifuged, re‐suspended and plated on chocolate agar plates in aerobic and anaerobic conditions for 7 days. Purity plating was performed and isolates identified by sequencing of the 16s rRNA gene.Sediment culture of MSU samples reveals a variety of species of bacteria in urine of both patients with OAB and controls. The average number of species, as identified by our methodology, is 5 per patient in both groups. In controls, the most notable species are Staphylococcus, Streptococcus and Lactobacillus. With increased age, these notable species decrease and other species such as Escherichia coli (E.coli), Campylobacter and Bifidobacterium increase in prevalence. Comparing the microbiome of age‐matched controls with that of patients with OAB, shows that the incidence of E.coli, Campylobacter, Enterococcus and Proteus are further increased.In an attempt to confirm that the bacteria observed was not a contamination artefact of using MSU samples, we performed similar analysis on CSU samples. Preliminary data indicates a trend for increases in the incidence of E.coli and Comamonadaceae and a decrease in Gardnerella and Staphylococcus in patients with OAB.Evidence presented here supports recent observation that the bladder has a microbiome and urine is not sterile. Furthermore, there appears to be differences in the bladder microbiome between patients with OAB and controls, and these differences become more apparent in the aged. A more thorough analysis of the microbiome, including antibiotic protection assays on each sample, is now under way.Support or Funding InformationKidney Research UK

Overactive Bladder (OAB) is an idiopathic condition, characterized by urgency, urinary frequency, and urgency incontinence, in the absence of routinely traceable urinary infection. We have described microscopic pyuria (≥10 wbc/μl) in patients suffering from the worst symptoms. It is established that inflammation is associated with increased ATP release from epithelial cells, and extracellular ATP originating from the urothelium following increased hydrostatic pressure is a mediator of bladder sensation. Here, using bladder biopsy samples, we have investigated urothelial ATP signaling in OAB patients with microscopic pyuria. Basal, but not stretch-evoked, release of ATP was significantly greater from the urothelium of OAB patients with pyuria than from non-OAB patients or OAB patients without pyuria (<10 wbc/μl). Basal ATP release from the urothelium of OAB patients with pyuria was inhibited by the P2 receptor antagonist suramin and abolished by the hemichannel blocker carbenoxolone, which differed from stretch-activated ATP release. Altered P2 receptor expression was evident in the urothelium from pyuric OAB patients. Furthermore, intracellular bacteria were visualized in shed urothelial cells from ∼80% of OAB patients with pyuria. These data suggest that increased ATP release from the urothelium, involving bacterial colonization, may play a role in the heightened symptoms associated with pyuric OAB patients.

Natural product inhibitors of AChE are of interest both because they offer promise as inexpensive drugs for symptomatic relief in Alzheimer's disease and because they may provide insights into the structural features of the AChE catalytic site. Hopeahainol A is an uncharged polyphenol AChE inhibitor from the stem bark of Hopea hainanensis with a constrained, partially dearomatized bicyclic core. Molecular modeling indicates that hopeahainol A binds at the entrance of the long but narrow AChE active site gorge because it is too bulky to be accommodated within the gorge without severe distortion of the gorge as depicted in AChE crystal structures. We conducted inhibitor competition experiments in which AChE inhibition was measured with hopeahainol A together with either edrophonium (which binds at the base of the gorge) or thioflavin T (which binds to the peripheral or P-site near the gorge mouth). The results agreed with the molecular modeling and indicated that hopeahainol A at lower concentrations (<200 μM) bound only to the P-site, as hopeahainol A and thioflavin T were unable to form a ternary complex with AChE while hopeahainol A and edrophonium did form a ternary complex with essentially no competition between them. Inhibition increased to a striking extent at higher concentrations of hopeahainol A, with plots analogous to classic Dixon plots showing a dependence on hopeahainol A concentrations to the third- or fourth order. The inhibition at higher hopeahainol A concentrations was completely reversed on dilution and blocked by bound edrophonium. We hypothesize that bound hopeahainol A induces conformational changes in the AChE active site that allow binding of additional hopeahainol A molecules, a phenomenon that would be unprecedented for a reversible inhibitor that apparently forms no covalent bonds with AChE.