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The final year of a biochemistry degree is usually a time to experience research. However, laboratory-based research projects were not possible during COVID-19. Instead, we used open datasets to provide computational research projects in metagenomics to biochemistry undergraduates (80 students with limited computing experience). We aimed to give the students a chance to explore any dataset, rather than use a small number of artificial datasets (~60 published datasets were used). To achieve this, we utilized Google Colaboratory (Colab), a virtual computing environment. Colab was used as a framework to retrieve raw sequencing data (analyzed with QIIME2) and generate visualizations. Setting up the environment requires no prior experience; all students have the same drive structure and notebooks can be shared (for synchronous sessions). We also used the platform to combine multiple datasets, perform a meta-analysis, and allowed the students to analyze large datasets with 1000s of subjects and factors. Projects that required increased computational resources were integrated with Google Cloud Compute. In future, all research projects can include some aspects of reanalyzing public data, providing students with data science experience. Colab is also an excellent environment in which to develop data skills in multiple languages (e.g., Perl, Python, Julia).

We apply the hit-to-lead ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent) and lead-optimization TIES (thermodynamic integration with enhanced sampling) methods to compute the binding free energies of a series of ligands at the A1and A2Aadenosine receptors, members of a subclass of the GPCR (G protein-coupled receptor) superfamily. Our predicted binding free energies, calculated using ESMACS, show a good correlation with previously reported experimental values of the ligands studied. Relative binding free energies, calculated using TIES, accurately predict experimentally determined values within a mean absolute error of approximately 1 kcal mol−1. Our methodology may be applied widely within the GPCR superfamily and to other small molecule–receptor protein systems.

The identification of strategies by which to increase the representation of women and increase diversity in STEM fields (science, technology, engineering and mathematics), including medicine, has been a pressing matter for global agencies including the European Commission, UNESCO and numerous international scientific societies. In my role as UCL training lead for CompBioMed, a European Commission Horizon 2020-funded Centre of Excellence in Computational Biomedicine (compbiomed.eu), and as Head of Teaching for Molecular Biosciences at UCL from 2010 to 2019, I have integrated research and teaching to lead the development of high-performance computing (HPC)-based education targeting medical students and undergraduate students studying biosciences in a way that is explicitly integrated into the existing university curriculum as a credit-bearing module. One version of the credit-bearing module has been specifically designed for medical students in their pre-clinical years of study and one of the unique features of the course is the integration of clinical and computational aspects, with students obtaining and processing clinical samples and then interrogating the results computationally using code that was ported to HPC at CompBioMed's HPC Facility core partners (EPCC (UK), SURFsara (The Netherlands) and the Barcelona Supercomputing Centre (Spain)). Another version of the credit-bearing module has, over the course of this project, evolved into a replacement for the third year research project course for undergraduate biochemistry, biotechnology and molecular biology students, providing students with the opportunity to design and complete an entire specialist research project from the formulation of experimental hypotheses to the investigation of these hypotheses in a way that involves the integration of experimental and HPC-based computational methodologies. Since 2017–2018, these UCL modules have been successfully delivered to over 350 students—a cohort with a demographic of greater than 50% female. CompBioMed's experience with these two university modules has enabled us to distil our methodology into an educational template that can be delivered at other universities in Europe and worldwide. This educational approach to training enables new communities of practice to effectively engage with HPC and reveals a means by which to improve the underrepresentation of women in supercomputing.

We describe a Xenopus P2Y receptor that shares only weak homology with members of the mammalian P2Y family, being most similar to human P2Y11. When activated by nucleotide analogs, it stimulates both calcium and cAMP mobilization pathways, a feature unique, among mammalian P2Y receptors, to P2Y11. Activity can be blocked by compounds known to act as antagonists of mammalian P2Y11. Genomic synteny between Xenopus and mammals suggests that the novel gene is a true ortholog of P2Y11. Xenopus P2Y11 is transcribed during embryonic development, beginning at gastrulation, and is enriched in the developing nervous system.

1 Many types of culture media contain a pH-sensitive dye. One commonly occurring dye, Phenol red sodium (Na+) salt, was tested for blocking activity at rat P2X1−4 receptors (P2X1−4Rs) expressed in Xenopus oocytes. 2 Phenol red Na+-salt antagonised adenosine 5′-triphosphate (ATP) responses at P2X1R (IC50, 3 μM) and, at higher concentrations, also blocked P2X2R and P2X3R. Phenol red Na+-salt, purified of lipophilic contaminants, blocked P2X1R and P2X3R by acting as an insurmountable antagonist. 3 Two lipophilic extracts of Phenol red antagonised ATP responses at P2XRs. Extract A was a potent antagonist at P2X1R (IC50, 1.4 μM), whereas extract B was a potent antagonist at P2X3R (IC50, 4.1 μM). A bisphenolic compound (RS151030) found in these extracts was a potent antagonist at P2X1R (IC50, 0.3 μM) and at P2X3R (IC50, 2.4 μM). 4 Phenolphthalein base was a potent irreversible antagonist at P2X1R (IC50, 1 μM), whereas Phenolphthalein K+-salt was 25-fold less potent here. 5 Phenolphthalein base was a reversible antagonist of ATP responses at rat P2X4R (IC50, 26 μM), whereas Phenolphthalein K+-salt was inactive. 6 Dimethyl sulphoxide (DMSO), used to dissolve lipophilic extracts, showed pharmacological activity by itself at rat P2X1R and P2X4R. 7 Thus, Phenol red and related compounds are antagonists at rat P2X1R, but are also active at other rat P2XRs. Phenolphthalein base is a newly identified, low potency antagonist of ATP responses at P2X4R. Culture media containing these red dyes should be used cautiously in future pharmacological studies of P2XRs. Also, wherever possible, the solvent DMSO should be used with caution. British Journal of Pharmacology (2005) 145, 313–322. doi:10.1038/sj.bjp.0706187

We have generated and characterised a clone of chicken DT40 lymphocytes stably transfected with the rat P2X(7) receptor (rP2X(7)). Successful transfection was confirmed by Western blotting. Under voltage clamp, P2X(7)-expressing cells responded to ATP and dibenzoyl-ATP (Bz-ATP) (a more potent P2X(7) receptor agonist) with a rapidly activating and sustained inward current. The EC(50) values for these agonists were 305 and 15 microM, respectively. Bz-ATP evoked Ca(2+) and Mn(2+) influx into transfected cells as determined by Fura-2 spectrofluorimetry. Responses to Bz-ATP were inhibited by pre-treatment of cells with oxidised ATP. Treatment of cells with Bz-ATP for up to 24hr produced time- and concentration-dependent cell death. This was associated with an increase in caspase-3-like activity, exposure of phosphatidylserine on the outside of cell membrane and DNA cleavage, indicating death by apoptosis. Pre-treatment with Z-VAD-fmk, a pan-caspase inhibitor, reduced the DNA fragmentation and phosphatidylserine externalisation, but did not affect overall rates of cell death at 24hr, implicating caspase-independent mechanisms. The properties of rP2X(7) receptors expressed in DT40 cells are similar to those described for other expression systems. Because DT40 cells lack functionally detectable endogenous P2 receptors and are highly amenable to genetic manipulation, stably transfected DT40 cells provide a novel and potentially useful model system in which to investigate the intracellular signal transduction pathways associated with P2X(7) receptor stimulation, in particular those involved in induction of cell death.

Rat P2X(1) and P2X(2) subunits were coexpressed in defolliculated Xenopus oocytes and the resultant P2X receptors studied under voltage-clamp conditions. Extracellular ATP elicited biphasic inward currents, involving an initial rapidly inactivating (P2X(1)-like) component and a later slowly inactivating (P2X(2)-like) component. The maximum amplitude of P2X(1)-like ATP responses was increased in some cells by lowering extracellular pH (from 7.5 to 6.5), whereas P2X(2)-like responses and those of homomeric rP2X(1) and rP2X(2) receptors were not changed by this treatment. Concentration-response (C/R) curves for ATP for pH-enhanced P2X(1)-like responses were biphasic, and clearly distinct from monophasic ATP C/R curves for homomeric rP2X(1) and rP2X(2) receptors. Under acidic (pH 5.5 and 6.5) and alkaline (pH 8.5) conditions, ATP C/R curves for P2X(1)-like responses showed increases in agonist potency and efficacy, compared with data at pH 7.5, but the same was not true of homomeric rP2X(1) and rP2X(2) receptors. ATP C/R curves for P2X(2)-like responses overlay C/R curves for homomeric rP2X(2) receptors, and determinations of agonist potency and efficacy were identical for P2X(2)-like and P2X(2) responses at all pH levels tested. Our results show that P2X(1)-like responses possessed the kinetics of homomeric P2X(1) receptors but an acid sensitivity different from homomeric P2X(1) and P2X(2) receptors. In contrast, the P2X(2)-like responses exactly matched the profile expected of homomeric P2X(2) receptors. Thus, coexpression of P2X(1) and P2X(2) subunits yielded a mixed population of homomeric and heteromeric P2X receptors, with a subpopulation of novel pH-sensitive P2X receptors showing identifiably unique properties that indicated the formation of heteromeric P2X(1/2) ion channels.

We have cloned and functionally characterised the mouse orthologue of the P2X4 receptor, mP2X4, and a splice variant of this receptor, mP2X4a. mP2X4 is 388 amino acids in length and shares 94% and 87% identity with the rat and human P2X4 receptors, respectively, while mP2X4a is 361 amino acids in length and lacks a 27-amino acid region in the extracellular domain corresponding to exon 6 of the known P2X receptor gene structures. When expressed in Xenopus laevis oocytes, mP2X4 produces a rapid inward current in response to ATP with an EC50 of 1.68+/-0.2 microM, consistent with the affinity of the rat and human P2X4 receptors for ATP. This agonist response is potentiated by the P2X receptor antagonists suramin, Reactive blue 2 and, over a limited concentration range, by PPADS. Although mP2X4a forms a poorly functional homomeric receptor, it appears able to interact with the full-length mP2X4 subunit to result in a functional channel with a reduced affinity for ATP. These results suggest a possible role for splice variants of P2X receptors in the formation of functional heteromeric ion channels.

Various adenosine analogues were tested at the adenosine A2B receptor. Agonist potencies were determined by measuring the cyclic AMP production in Chinese Hamster Ovary cells expressing human A2B receptors. 5'-N-Substituted carboxamidoadenosines were most potent. 5'-N-Ethylcarboxamidoadenosine (NECA) was most active with an EC50 value of 3.1 microM. Other ribose modified derivatives displayed low to negligible activity. Potency was reduced by substitution on the exocyclic amino function (N6) of the purine ring system. The most active N6-substituted derivative N6-methyl-NECA was 5 fold less potent than NECA. C8- and most C2-substituted analogues were virtually inactive. 1-Deaza-analogues had a reduced potency, 3- and 7-deazaanalogues were not active.

The gene for the stimulatory G protein-coupled human A2a adenosine receptor was isolated and sequence analysis revealed two exons that are interrupted by an intron of approximately 6.4 kb. An intron is located in the same region in the human A1 and A2b adenosine receptor genes. Comparison of the A2a genomic and cDNA sequences reveals two nucleotide differences in the coding region and the presence of an aberrant sequence in the 5'208 base pairs of the A2a cDNA including a polymorphism in the third base of codon Tyr-361 and Gly codon which was always detected at residue 392, indicated that the Arg codon present in the cDNA may be an artifact. Fluorescent in situ hybridization and PCR analysis of human-hamster hybrid cell panels shows that the A2a receptor gene is localized to chromosome 22q11.2. This is in contrast with previous reports (subsequently retracted) which mapped the A2a gene to chromosome 11q11-13.

The A1 adenosine receptor is a member of the seven-transmembrane G protein-coupled, receptor superfamily. This receptor binds the purine nucleoside adenosine with high affinity and inhibits the activity of adenylate cyclase. We have used site-directed mutagenesis and functional expression studies to examine the role of the threonine residue, located at position 277 in transmembrane domain VII of the human A1 receptor. Mutation of Thr-277 to either serine or alanine resulted in the expression of receptors that had essentially no change in binding affinity for the A1 selective antagonist 8-cyclo-pentyl-1,3-dipropylxanthine. Mutation of Thr-277 to serine resulted in modest (4.4-8.6-fold) but significant increases in the observed Ki values for three adenosine agonists, namely N-(1-methyl-2-phenethyl)adenosine (R-PIA or S-PIA) and 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-L- ribofuranuronamide) (NECA). However, mutation of Thr-277 to alanine resulted in no significant changes in the Ki for R-PIA or S-PIA but did result in a highly significant 437-fold increase in the Ki for NECA. This demonstrates that the hydroxyl moiety of Thr-277 mediates agonist but not antagonist binding and, more specifically, that this residue forms a probable molecular contact site with the 5' substitution found in NECA.

Bio-electrospraying (BES) is a method for directly jetting living cells under conditions that allow their distribution in the x, y, and z axes. Previous work has been focused on achieving jetting in stable cone-jet mode, which is required for precision placement, and these studies have demonstrated that there are no significant effects of bio-electrospraying on cell morphology or viability. In this work, we examine the biological properties of bio-electrosprayed cells using assays of cellular function that range from the molecular level through to integrated cellular systems, and include proteomics, signal transduction, cell growth and proliferation, and the characterisation of apoptotic blebs. From these molecular methods, we have determined that bio-electrospraying, under the electric field conditions used to achieve stable cone-jet mode, causes no alterations to the biological properties and function of the cells being jetted. Bio-electrosprayed and control cells had similar viability, proliferation properties and virtually indistinguishable cell cycle profiles. The biophysical properties of large conducting (BK) potassium channels were unchanged, as were the pharmacological responses of the endogenous muscarinic and exogenous P2Y(11) receptors, both of which are cell surface receptors of the 7TM superfamily. Proteomic analyses revealed that although three proteins had subtle differences in expression level between bio-electrosprayed and control cells, none of these fold differences was above the 1.5-fold cut-off threshold required for further analyses. These findings support the further development of bio-electrosprays as a viable technology for a wide diversity of tissue engineering, regenerative biology, advanced cellular therapeutics and medicinal applications, having significance in the clinic.

Using the sequence conservation in the G protein-coupled receptor superfamily, we have isolated an adenosine A1 receptor cDNA from a human hippocampal cDNA library by homology screening. When expressed in mammalian CHO.K1 cells, the protein encoded by this cDNA binds the A1-specific antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) with high affinity (Kd = 0.56 +/- 0.11 nM) and, functionally, is able to inhibit cAMP production upon receptor activation with the A1-specific agonist N6-cyclopentyladenosine (CPA) (> 80% inhibition at 10(-7) M CPA). The binding and functional characteristics of the expressed cDNA demonstrate that we have isolated a human brain adenosine receptor cDNA of the A1 subtype.

Electrospraying, or electrohydrodynamic jetting, is one of several jet-based technologies being explored to process living biological organisms. One of the key advantages of electrospraying is its ability to deposit advanced materials with high resolution that cannot be obtained with other competing technologies, such as ink-jet printing. However, to generate a controlled droplet size distribution in the micrometre range necessary for precision drop and placement of materials requires jetting in stable cone-jet mode. In this paper, we describe the experimental set-up and conditions by which electrospray jetting in stable cone-jet is achieved and use this methodology to process a highly concentrated biological suspension having 10(7) cells ml(-1), the highest cellular loading processed to this day by a jetting approach in this jet based category. The areas of study to which this technology may be applied span the physical and the life sciences.

Silicon-based microphysiometry, measuring extracellular acidification rate of cells in culture, demonstrated that a series of diimidazo[1,2-c:4',5'-e]pyrimidines were agonists at the human adenosine A1 receptor. 5-amino-7,8-dihydro-3-ribofuranose-8-(R)-(phenyl)-3H-diimidazo [1,2-c:4',5'-e]pyrimidine (2a) had an EC50 of 100 microM and reached 90% of the Emax produced by R-PIA.

In the past five years, an extended series (P2Y1-n) of metabotropic nucleotide (P2) receptors has been cloned from vertebrate tissues; these receptors are activated by either ATP or UTP, or both nucleotides. While certain cloned P2Y receptors appear to correspond functionally to particular native P2 receptor phenotypes, such pharmacological phenotypes could be explained by either a combination of several members of the P2Y1-n series being coexpressed in the same tissue or the existence of novel, uncloned P2Y subtypes. Here, Brian King, Andrea Townsend-Nicholson and Geoffrey Burnstock review recent findings on the matter of pharmacological relationships between native P2 and cloned P2Y receptors.

A thermodynamic analysis of the binding of a full agonist (N6-cyclopentyladenosine), a partial agonist (8-butylamino-N6-cyclopentyladenosine) and an antagonist (8-cyclopentyltheophylline) to human wild-type and mutant (mutation of a threonine (Thr) to an alanine (Ala) residue at position 277) adenosine A1 receptors expressed on Chinese hamster ovary (CHO) cells, and to rat brain adenosine A1 receptors was undertaken. The thermodynamic parameters deltaGo (standard free energy), deltaHo (standard enthalpy) and deltaSo (standard entropy) of the binding equilibrium to rat brain receptors were determined by means of affinity measurements carried out at four different temperatures (0, 10, 20 and 25 degrees) and van't Hoff plots. Two temperatures (0 and 25 degrees) were considered for human receptors. Affinity constants were obtained from inhibition assays on membrane preparations of rat brain and CHO cells by use of the antagonist [3H]1,3-dipropyl-8-cyclopentylxanthine ([3H]DPCPX) as selective adenosine A1 receptor radioligand. As for rat brain receptors, full agonist binding was totally entropy driven, whereas antagonist binding was essentially enthalpy driven. Partial agonist binding appeared both enthalpy and entropy driven. As for human receptors, full agonist affinity was highly dependent on the presence of Thr277. Moreover, affinity to both wild-type and mutant receptors was enhanced by temperature increase, suggesting a totally entropy-driven binding. Antagonist binding did not depend on the presence of Thr277. Antagonist affinity decreased with an increase in temperature, suggesting a mainly enthalpy-driven binding. Partial agonist binding was significantly dependent on the presence of Thr277 at 25 degrees, whereas such a dependence was not evident at 0 degrees. It is concluded that Thr277 contributes only to the binding of adenosine derivatives and that its role changes drastically with the receptor conformation and with the type of agonist (full or partial) interacting with the adenosine A1 receptors.

1. The effect of adenosine A1-receptor and P2-purinoceptor agonists on [3H]-inositol phosphate accumulation has been investigated in CHO-K1 cells transfected with the human adenosine A1-receptor. 2. Adenosine receptor agonists stimulated [3H]-inositol phosphate accumulation in CHO-K1 cells with a rank potency order of N6-cyclopentyladenosine (CPA) > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-chloroadenosine > N6-2-(4-aminophenyl) ethyladenosine (APNEA). The responses to both CPA and APNEA were antagonized by the A1 selective antagonist, 1,3-dipropylcyclopentylxanthine (DPCPX) yielding KD values of 1.2 nM and 4.3 nM respectively. 3. ATP, UTP and ATP gamma S were also able to stimulate [3H]-inositol phosphate accumulation in these cells with EC50 values of 1.9 microM, 1.3 microM and 5.0 microM respectively. 2-Methyl-thio-ATP was a weak agonist of this response (EC50 > 100 microM). 4. The [3H]-inositol phosphate response to CPA was completely attenuated by pertussis toxin treatment (24 h; 100 ng ml-1). In contrast, the responses to ATP, UTP and ATP gamma S were only reduced by circa 30% in pertussis toxin-treated cells. 5. The simultaneous addition of CPA and either ATP, UTP or ATP gamma S produced a large augmentation of [3H]-inositol phospholipid hydrolysis. This was due to an increase in the maximal response and was significantly greater than the predicted additive response for activation of these two receptor systems. The synergy was not observed in pertussis toxin-treated cells. 6. No synergy was observed between the [3H]-inositol phosphate responses to histamine and ATP in CHO-K1 cells transfected with the bovine histamine H1-receptor. In these cells the response to histamine was completely resistant to inhibition by pertussis toxin treatment. 7. This study provides a clear demonstration of a synergy between pertussis toxin-sensitive and insensitive receptor systems in a model cell system which is an ideal host for transfected cDNA sequences. This model system should provide a unique opportunity to unravel the mechanisms underlying this example of receptor cross-talk involving phospholipase C.