Estimation of model accuracy in CASP15 using the ModFOLDdock server.

In CASP15, there was a greater emphasis on multimeric modeling than in previous experiments, with assembly structures nearly doubling in number (41 up from 22) since the previous round. CASP15 also included a new estimation of model accuracy (EMA) category in recognition of the importance of objective quality assessment (QA) for quaternary structure models. ModFOLDdock is a multimeric model QA server developed by the McGuffin group at the University of Reading, which brings together a range of single-model, clustering, and deep learning methods to form a consensus of approaches. For CASP15, three variants of ModFOLDdock were developed to optimize for the different facets of the quality estimation problem. The standard ModFOLDdock variant produced predicted scores optimized for positive linear correlations with the observed scores. The ModFOLDdockR variant produced predicted scores optimized for ranking, that is, the top-ranked models have the highest accuracy. In addition, the ModFOLDdockS variant used a quasi-single model approach to score each model on an individual basis. The scores from all three variants achieved strongly positive Pearson correlation coefficients with the CASP observed scores (oligo-lDDT) in excess of 0.70, which were maintained across both homomeric and heteromeric model populations. In addition, at least one of the ModFOLDdock variants was consistently ranked in the top two methods across all three EMA categories. Specifically, for overall global fold prediction accuracy, ModFOLDdock placed second and ModFOLDdockR placed third; for overall interface quality prediction accuracy, ModFOLDdockR, ModFOLDdock, and ModFOLDdockS were placed above all other predictor methods, and ModFOLDdockR and ModFOLDdockS were placed second and third respectively for individual residue confidence scores. The ModFOLDdock server is available at: https://www.reading.ac.uk/bioinf/ModFOLDdock/. ModFOLDdock is also available as part of the MultiFOLD docker package: https://hub.docker.com/r/mcguffin/multifold.

Improvement of protein tertiary and quaternary structure predictions using the ReFOLD refinement method and the AlphaFold2 recycling process.

Motivation: The accuracy gap between predicted and experimental structures has been significantly reduced following the development of AlphaFold2 (AF2). However, for many targets, AF2 models still have room for improvement. In previous CASP experiments, highly computationally intensive MD simulation-based methods have been widely used to improve the accuracy of single 3D models. Here, our ReFOLD pipeline was adapted to refine AF2 predictions while maintaining high model accuracy at a modest computational cost. Furthermore, the AF2 recycling process was utilized to improve 3D models by using them as custom template inputs for tertiary and quaternary structure predictions. Results: According to the Molprobity score, 94% of the generated 3D models by ReFOLD were improved. AF2 recycling showed an improvement rate of 87.5% (using MSAs) and 81.25% (using single sequences) for monomeric AF2 models and 100% (MSA) and 97.8% (single sequence) for monomeric non-AF2 models, as measured by the average change in lDDT. By the same measure, the recycling of multimeric models showed an improvement rate of as much as 80% for AF2-Multimer (AF2M) models and 94% for non-AF2M models. Availability and implementation: Refinement using AlphaFold2-Multimer recycling is available as part of the MultiFOLD docker package (https://hub.docker.com/r/mcguffin/multifold). The ReFOLD server is available at https://www.reading.ac.uk/bioinf/ReFOLD/ and the modified scripts can be downloaded from https://www.reading.ac.uk/bioinf/downloads/. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Prediction of protein structures, functions and interactions using the IntFOLD7, MultiFOLD and ModFOLDdock servers.

The IntFOLD server based at the University of Reading has been a leading method over the past decade in providing free access to accurate prediction of protein structures and functions. In a post-AlphaFold2 world, accurate models of tertiary structures are widely available for even more protein targets, so there has been a refocus in the prediction community towards the accurate modelling of protein-ligand interactions as well as modelling quaternary structure assemblies. In this paper, we describe the latest improvements to IntFOLD, which maintains its competitive structure prediction performance by including the latest deep learning methods while also integrating accurate model quality estimates and 3D models of protein-ligand interactions. Furthermore, we also introduce our two new server methods: MultiFOLD for accurately modelling both tertiary and quaternary structures, with performance which has been independently verified to outperform the standard AlphaFold2 methods, and ModFOLDdock, which provides world-leading quality estimates for quaternary structure models. The IntFOLD7, MultiFOLD and ModFOLDdock servers are available at: https://www.reading.ac.uk/bioinf/.

Computational Methods for the Elucidation of Protein Structure and Interactions.

Biologists are increasingly aware of the importance of protein structure in revealing function. The computational tools now exist which allow researchers to model unknown proteins simply on the basis of their primary sequence. However, for the non-specialist bioinformatician, there is a dazzling array of terminology, acronyms, and competing computer software available for this process. This review is intended to highlight the key stages of computational protein structure prediction, as well as explain the reasons behind some of the procedures and list some established workarounds for common pitfalls. Thereafter follows a review of five one-stop servers for start-to-finish structure prediction.

Functionalized lipid nanoparticles for subcutaneous administration of mRNA to achieve systemic exposures of a therapeutic protein.

Lipid nanoparticles (LNPs) are the most clinically advanced delivery system for RNA-based drugs but have predominantly been investigated for intravenous and intramuscular administration. Subcutaneous administration opens the possibility of patient self-administration and hence long-term chronic treatment that could enable messenger RNA (mRNA) to be used as a novel modality for protein replacement or regenerative therapies. In this study, we show that subcutaneous administration of mRNA formulated within LNPs can result in measurable plasma exposure of a secreted protein. However, subcutaneous administration of mRNA formulated within LNPs was observed to be associated with dose-limiting inflammatory responses. To overcome this limitation, we investigated the concept of incorporating aliphatic ester prodrugs of anti-inflammatory steroids within LNPs, i.e., functionalized LNPs to suppress the inflammatory response. We show that the effectiveness of this approach depends on the alkyl chain length of the ester prodrug, which determines its retention at the site of administration. An unexpected additional benefit to this approach is the prolongation observed in the duration of protein expression. Our results demonstrate that subcutaneous administration of mRNA formulated in functionalized LNPs is a viable approach to achieving systemic levels of therapeutic proteins, which has the added benefits of being amenable to self-administration when chronic treatment is required.

Extracellular vesicles induce minimal hepatotoxicity and immunogenicity.

Extracellular vesicles (EVs) mediate cellular communication through the transfer of active biomolecules, raising interest in using them as biological delivery vehicles for therapeutic drugs. For drug delivery applications, it is important to understand the intrinsic safety and toxicity liabilities of EVs. Nanoparticles, including EVs, typically demonstrate significant accumulation in the liver after systemic administration in vivo. We confirmed uptake of EVs derived from Expi293F cells into HepG2 cells and did not detect any signs of hepatotoxicity measured by cell viability, functional secretion of albumin, plasma membrane integrity, and mitochondrial and lysosomal activity even at high exposures of up to 5 × 1010 EVs per mL. Whole genome transcriptome analysis was used to measure potential effects on the gene expression in the recipient HepG2 cells at 24 h following exposure to EVs. Only 0.6% of all genes were found to be differentially expressed displaying less than 2-fold expression change, with genes related to inflammation or toxicity being unaffected. EVs did not trigger any proinflammatory cytokine response in HepG2 cells. However, minor changes were noted in human blood for interleukin (IL)-8, IL-6, and monocyte chemotactic protein 1 (MCP-1). Administration of 5 × 1010 Expi293F-derived EVs to BALB/c mice did not result in any histopathological changes or increases of liver transaminases or cytokine levels, apart from a modest increase in keratinocyte chemoattractant (KC). The absence of any significant toxicity associated with EVs in vitro and in vivo supports the prospective use of EVs for therapeutic applications and for drug delivery.

2'-O-(2-Methoxyethyl) Nucleosides Are Not Phosphorylated or Incorporated Into the Genome of Human Lymphoblastoid TK6 Cells.

Nucleoside analogs with 2'-modified sugar moieties are often used to improve the RNA target affinity and nuclease resistance of therapeutic oligonucleotides in preclinical and clinical development. Despite their enhanced nuclease resistance, oligonucleotides could slowly degrade releasing nucleoside analogs that have the potential to become phosphorylated and incorporated into cellular DNA and RNA. For the first time, the phosphorylation and DNA/RNA incorporation of 2'-O-(2-methoxyethyl) (2'-O-MOE) nucleoside analogs have been investigated. Using liquid chromatography/tandem mass spectrometry, we showed that enzymes in the nucleotide salvage pathway including deoxycytidine kinase (dCK) and thymidine kinase (TK1) displayed poor reactivity toward 2'-O-MOE nucleoside analogs. On the other hand, 2'-fluoro (F) nucleosides, regardless of the nucleobase, were efficiently phosphorylated to their monophosphate forms by dCK and TK1. Consistent with their efficient phosphorylation by dCK and TK1, 2'-F nucleoside analogs were incorporated into cellular DNA and RNA while no incorporation was detected with 2'-O-MOE nucleoside analogs. In conclusion, these data suggest that the inability of dCK and TK1 to create the monophosphates of 2'-O-MOE nucleoside analogs reduces the risk of their incorporation into cellular DNA and RNA.

Predicting Drug Safety and Communicating Risk: Benefits of a Bayesian Approach.

Drug toxicity is a major source of attrition in drug discovery and development. Pharmaceutical companies routinely use preclinical data to predict clinical outcomes and continue to invest in new assays to improve predictions. However, there are many open questions about how to make the best use of available data, combine diverse data, quantify risk, and communicate risk and uncertainty to enable good decisions. The costs of suboptimal decisions are clear: resources are wasted and patients may be put at risk. We argue that Bayesian methods provide answers to all of these problems and use hERG-mediated QT prolongation as a case study. Benefits of Bayesian machine learning models include intuitive probabilistic statements of risk that incorporate all sources of uncertainty, the option to include diverse data and external information, and visualizations that have a clear link between the output from a statistical model and what this means for risk. Furthermore, Bayesian methods are easy to use with modern software, making their adoption for safety screening straightforward. We include R and Python code to encourage the adoption of these methods.

A role for brain size and cognition in food webs.

Predators tend to be large and mobile, enabling them to forage in spatially distinct food web compartments (e.g. littoral and pelagic aquatic macrohabitats). This feature can stabilise ecosystems when predators are capable of rapid behavioural response to changing resource conditions in distinct habitat compartments. However, what provides this ability to respond behaviourally has not been quantified. We hypothesised that predators require increased cognitive abilities to occupy their position in a food web, which puts pressure to increase brain size. Consistent with food web theory, we found that fish relative brain size increased with increased ability to forage across macrohabitats and increased relative trophic positions in a lacustrine food web, indicating that larger brains may afford the cognitive capacity to exploit various habitats flexibly, thus contributing to the stability of whole food webs.

Food Web Structure Shapes the Morphology of Teleost Fish Brains.

Previous work showed that teleost fish brain size correlates with the flexible exploitation of habitats and predation abilities in an aquatic food web. Since it is unclear how regional brain changes contribute to these relationships, we quantitatively examined the effects of common food web attributes on the size of five brain regions in teleost fish at both within-species (plasticity or natural variation) and between-species (evolution) scales. Our results indicate that brain morphology is influenced by habitat use and trophic position, but not by the degree of littoral-pelagic habitat coupling, despite the fact that the total brain size was previously shown to increase with habitat coupling in Lake Huron. Intriguingly, the results revealed two potential evolutionary trade-offs: (i) relative olfactory bulb size increased, while relative optic tectum size decreased, across a trophic position gradient, and (ii) the telencephalon was relatively larger in fish using more littoral-based carbon, while the cerebellum was relatively larger in fish using more pelagic-based carbon. Additionally, evidence for a within-species effect on the telencephalon was found, where it increased in size with trophic position. Collectively, these results suggest that food web structure has fundamentally contributed to the shaping of teleost brain morphology.

Exenatide exerts a PKA-dependent positive inotropic effect in human atrial myocardium: GLP-1R mediated effects in human myocardium.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are a rapidly growing class of drugs developed for treating type-2 diabetes mellitus. Patients with diabetes carry an up to 5-fold greater mortality risk compared to non-diabetic patients, mainly as a result of cardiovascular diseases. Although beneficial cardiovascular effects have been reported, exact mechanisms of GLP-1R-agonist action in the heart, especially in human myocardium, are poorly understood. The effects of GLP-1R-agonists (exenatide, GLP-1(7-36)NH2, PF-06446009, PF-06446667) on cardiac contractility were tested in non-failing atrial and ventricular trabeculae from 72 patients. The GLP-1(7-36)NH2 metabolite, GLP-1(9-36)NH2, was also examined. In electrically stimulated trabeculae, the effects of compounds on isometric force were measured in the absence and presence of pharmacological inhibitors of signal transduction pathways. The role of ß-arrestin signaling was examined using a ß-arrestin partial agonist, PF-06446667. Expression levels were tested by immunoblots. Translocation of GLP-1R downstream molecular targets, Epac2, GLUT-1 and GLUT-4, were assessed by fluorescence microscopy. All tested GLP-1R-agonists significantly increased developed force in human atrial trabeculae, whereas GLP-1(9-36)NH2 had no effect. Exendin(9-39)NH2, a GLP-1R-antagonist, and H-89 blunted the inotropic effect of exenatide. In addition, exenatide increased PKA-dependent phosphorylation of phospholamban (PLB), GLUT-1 and Epac2 translocation, but not GLUT-4 translocation. Exenatide failed to enhance contractility in ventricular myocardium. Quantitative real-time PCR (qRT-PCR) revealed a significant higher GLP-1R expression in the atrium compared to ventricle. Exenatide increased contractility in a dose-dependent manner via GLP-1R/cAMP/PKA pathway and induced GLUT-1 and Epac2 translocation in human atrial myocardium, but had no effect in ventricular myocardium. Therapeutic use of GLP-1R-agonists may therefore impart beneficial effects on myocardial function and remodelling.

Using ecology to inform physiology studies: implications of high population density in the laboratory.

Conspecific density is widely recognized as an important ecological factor across the animal kingdom; however, the physiological impacts are less thoroughly described. In fact, population density is rarely mentioned as a factor in physiological studies on captive animals and, when it is infrequently addressed, the animals used are reared and housed at densities far above those in nature, making the translation of results from the laboratory to natural systems difficult. We survey the literature to highlight this important ecophysiological gap and bring attention to the possibility that conspecific density prior to experimentation may be a critical factor influencing results. Across three taxa: mammals, birds, and fish, we present evidence from ecology that density influences glucocorticoid levels, immune function, and body condition with the intention of stimulating discussion and increasing consideration of population density in physiology studies. We conclude with several directives to improve the applicability of insights gained in the laboratory to organisms in the natural environment.

Pyrimido[4,5-d]azepines as potent and selective 5-HT2C receptor agonists: design, synthesis, and evaluation of PF-3246799 as a treatment for urinary incontinence.

New pyrimido[4,5-d]azepines 7 are disclosed as potent 5-HT(2C) receptor agonists. A preferred example, 7b had minimal activation at either the 5-HT(2A) or 5-HT(2B) receptors combined with robust efficacy in a preclinical canine model of stress urinary incontinence (SUI) and attractive pharmacokinetic and safety properties. Based on this profile, 7b (PF-3246799) was identified as a candidate for clinical development for the treatment of SUI. In addition, it proved to be critical to build an understanding of the translation between recombinant cell-based systems, native tissue preparations and in vivo preclinical models. This was a significant undertaking and proved to be crucial in compound selection.

Potentiation of reflex erectile responses in the anaesthetized rat by the selective melanocortin receptor 4 agonist MB243.

OBJECTIVE: To assess the potentiation of erectile responses induced by electrical stimulation (ES) of the dorsal penile nerve (DPN) in the urethane-anaesthetized rat by the selective melanocortin receptor 4 agonist MB243. MATERIALS AND METHODS: Intracavernosal and blood pressures (ICP and BP, respectively) were monitored in urethane-anaesthetized rats after complete spinal cord transection at the thoracic level. Erectile responses were induced by ES of the DPN (train of square wave pulses of 1 ms and 6 V for 20 s at 1, 2 and 5 Hz) after i.v. injection of either saline or MB243, 3 mg/kg, in two groups of six rats. The maximal and mean ICP, and the area under the curve (AUC) of the ICP response, corrected for the corresponding BP, were measured and used as an index of erectile function (ICPmax/BP, ICPmean/BP and AUC/BP, respectively). RESULTS: MB243 increased the number of spontaneous erections between the injection and the first ES when compared with the vehicle group, but this difference was not statistically significant. ES of the DPN induced frequency-dependent erectile responses, the mean (sem) ICPmean/BP was 0.26 (0.02), 0.34 (0.04) and 0.39 (0.05) after administration of saline (vehicle) at 1, 2 and 5 Hz, respectively. All the variables, except the ICPmax/BP at 5 Hz, were significantly increased in the group injected with MB243 when compared with the vehicle group (P < 0.05 for ICPmax/BP and ICPmean/BP; P < 0.01 for AUC/BP). The AUC/BP showed the greatest increases of (+79%, +60% and +44% at 1, 2 and 5 Hz, respectively) in the group injected with MB243 compared with the vehicle group. CONCLUSION: Erectile responses induced by ES of the DPN in spinalized, urethane-anaesthetized rat are suitable for evaluating the proerectile facilitator activity of selective peripherally restricted melanocortin receptor 4 agonists. This model represents a valuable alternative to the classically used cavernous/pelvic nerve stimulated model.

Does nitric oxide allow endothelial cells to sense hypoxia and mediate hypoxic vasodilatation? In vivo and in vitro studies.

Hypoxia-evoked vasodilatation is a fundamental regulatory mechanism that is often attributed to adenosine. The identity of the O(2) sensor is unknown. Nitric oxide (NO) inhibits endothelial mitochondrial respiration and ATP generation by competing with O(2) for its binding site on cytochrome oxidase. We proposed that in vivo this interaction allows endothelial cells to release adenosine when O(2) tension falls or NO concentration increases. Using anaesthetised rats, we confirmed that the increase in femoral vascular conductance (FVC, hindlimb vasodilatation) evoked by systemic hypoxia is attenuated by NO synthesis blockade with L-NAME, but restored when baseline FVC is restored by infusion of NO donor. This "restored" hypoxic response, like the control hypoxic response, is inhibited by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Similarly, the FVC increase evoked by adenosine infusion was attenuated by L-NAME but restored by infusion of NO donor. However, when baseline FVC was restored after L-NAME with 8-bromo-cGMP, the FVC increase evoked by adenosine infusion was restored, but not in response to systemic hypoxia, suggesting that adenosine was no longer released by hypoxia. Infusion of NO donor at a given rate after treatment with L-NAME evoked a greater FVC increase during systemic hypoxia than during normoxia, both responses being reduced by 8-cyclopentyl-1,3-dipropylxanthine. Finally, both bradykinin and NO donor released adenosine from superfused endothelial cells in vitro; L-NAME attenuated only the former response. We propose that in vivo, shear-released NO increases the apparent K(m) of endothelial cytochrome oxidase for O(2), allowing the endothelium to act as an O(2) sensor, releasing adenosine in response to moderate falls in O(2).