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Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y1R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y1R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice. Supplementary Information The online version contains supplementary material available at 10.1007/s11302-021-09792-9.

During the third wave of the COVID-19 (coronavirus disease 2019) pandemic in Germany, free SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) point-of-care (PoC) antigen tests were offered to citizens at least once a week to prevent spreading by asymptomatic infected individuals. This study investigated user groups, timing, frequency, and test center locations in a typical medium-sized European city. We analyzed 27,369 pseudonymized datasets from eight centers over 12 weeks. Those were evaluated according to age, residence, appointment, and potential repeated test occurrence. The centers were visited by different groups; some centers were preferred by a predominantly younger demographic, whereas a mobile option attracted an older age group by reaching districts with few other testing possibilities. Elderly individuals were tested more spontaneously than younger individuals, and a test center at a ‘park and ride’ had more spontaneous visitors from outside of the city compared to other test locations. Only a small proportion of less than 4% came for testing more than five times. To preferably address many people for voluntary antigen testing, it is crucial to offer different test opportunities accounting for individual behavioral patterns, despite this requiring more complex and costly design than conventional forms.

Reconciliation in South Africa is often taken to mean the creation of culturally diverse communities. In reality, though, the multicultural often turns out to be multiracial only with People of Colour being included in White-dominated spaces. Likewise, socio-economic transformation means raising people’s chances to attain a living standard more equal to that of the bulk of the White population. In both cases, the strong position of White people in sociocultural and socio-economic terms remains largely untouched. Hence the calls for decolonisation which seem to render the reconciliation discourse dispensable. Vulnerability by White South Africans is proposed as an alternative response to ongoing inequalities which – it is suggested – could contribute to both decolonisation and reconciliation on an interpersonal level. Likely objections to such a proposal are considered before making a case for vulnerability as an appropriate Christian way of living, particularly in the context of former settler colonialism.

Background Tourniquets used for peripheral venous vascular access such as blood sampling are regularly contaminated in clinical routine. Although most contaminations are harmless, some pose a possible risk for infection. To improve peripheral venous access infection control standards, tourniquets with no or as few as possible bacterial burden should be used. Conventional tourniquets can be reprocessed by autoclaving or by incubating in disinfectants. However, both methods are time-consuming and not suitable for immediate use between patients. In contrast, silicone tourniquets can be quickly and simply reprocessed with wipe disinfection. In vitro studies from the manufacturer have demonstrated reduced bacterial contamination on silicone tourniquets after usage compared to conventional tourniquets. This study aims to independently investigate the bacterial load on both types of tourniquets in clinical routine. Methods In a first trial, new conventional and silicon tourniquets were used for blood sampling in one facility with strict guidelines for reprocessing (after each patient or not at all) for 1 day and tested for bacterial contamination. In a second trial, new tourniquets were used in four facilities while the mode and frequency of tourniquets’ reprocessing was defined individually by each facility. The number of treated patients, mode and frequency of reprocessing and other relevant handling measures were documented. Results Under controlled conditions, with strictly specified reprocessing, slightly fewer bacteria were found on silicone than on conventional tourniquets. In routine clinical practice the reprocessing frequency was not higher for silicone tourniquets in practice. Yet, in all four facilities, there were significantly fewer bacteria found on silicone than on conventional tourniquets. Conclusion Although tourniquets are classified as non-critical medical devices, results show – together with benefits of faster and easier reprocessing – that silicone tourniquets can improve infection control of venous vascular access.

In response to dehydration, humans experience thirst. This subjective state is fundamental to survival as it motivates drinking, which subsequently corrects the fluid deficit. To elicit thirst, previous studies have manipulated blood chemistry to produce a physiological thirst stimulus. In the present study, we investigated whether a physiological stimulus is indeed required for thirst to be experienced. Functional MRI (fMRI) was used to scan fully hydrated participants while they imagined a state of intense thirst and while they imagined drinking to satiate thirst. Subjective ratings of thirst were significantly higher for imagining thirst compared with imagining drinking or baseline, revealing a successful dissociation of thirst from underlying physiology. The imagine thirst condition activated brain regions similar to those reported in previous studies of physiologically evoked thirst, including the anterior midcingulate cortex (aMCC), anterior insula, precentral gyrus, inferior frontal gyrus, middle frontal gyrus, and operculum, indicating a similar neural network underlies both imagined thirst and physiologically evoked thirst. Analogous brain regions were also activated during imagined drinking, suggesting the neural representation of thirst contains a drinking-related component. Finally, the aMCC showed an increase in functional connectivity with the insula during imagined thirst relative to imagined drinking, implying functional connectivity between these two regions is needed before thirst can be experienced. As a result of these findings, this study provides important insight into how the neural representation of subjective thirst is generated and how it subsequently motivates drinking behavior.

Summary Obesity is a major driver of cancer, especially hepatocellular carcinoma (HCC). The prevailing view is that non-alcoholic steatohepatitis (NASH) and fibrosis or cirrhosis are required for HCC in obesity. Here, we report that NASH and fibrosis and HCC in obesity can be dissociated. We show that the oxidative hepatic environment in obesity inactivates the STAT-1 and STAT-3 phosphatase T cell protein tyrosine phosphatase (TCPTP) and increases STAT-1 and STAT-3 signaling. TCPTP deletion in hepatocytes promoted T cell recruitment and ensuing NASH and fibrosis as well as HCC in obese C57BL/6 mice that normally do not develop NASH and fibrosis or HCC. Attenuating the enhanced STAT-1 signaling prevented T cell recruitment and NASH and fibrosis but did not prevent HCC. By contrast, correcting STAT-3 signaling prevented HCC without affecting NASH and fibrosis. TCPTP-deletion in hepatocytes also markedly accelerated HCC in mice treated with a chemical carcinogen that promotes HCC without NASH and fibrosis. Our studies reveal how obesity-associated hepatic oxidative stress can independently contribute to the pathogenesis of NASH, fibrosis, and HCC., Graphical Abstract, Highlights • Obesity promotes hepatic STAT-1 and STAT-3 signaling • Obesity promotes STAT-1-dependent T cell-infiltration, NASH, and fibrosis • Obesity promotes NASH-independent STAT-3-dependent HCC, Independent pathways contribute to development of NASH and hepatocellular carcinoma, disentangling the pathologies and providing insight for cancer treatments distinct from liver disease.

Homomeric P2X3 receptors are present in sensory ganglia and participate in pain perception. Amino acid (AA) residues were replaced in the four supposed nucleotide binding segments (NBSs) of the human (h) P2X3 receptor by alanine, and these mutants were expressed in HEK293 cells and Xenopus laevis oocytes. Patch clamp and two-electrode voltage clamp measurements as well as the Ca(2+) imaging technique were used to compare the concentration-response curves of the selective P2X1,3 agonist α,β-methylene ATP obtained at the wild-type P2X3 receptor and its NBS mutants. Within these NBSs, certain Gly (Gly-66), Lys (Lys-63, Lys-176, Lys-284, Lys-299), Asn (Asn-177, Asn-279), Arg (Arg-281, Arg-295), and Thr (Thr-172) residues were of great importance for a full agonist response. However, the replacement of further AAs in the NBSs by Ala also appeared to modify the amplitude of the current and/or [Ca(2+)](i) responses, although sometimes to a minor degree. The agonist potency decrease was additive after the simultaneous replacement of two adjacent AAs by Ala (K65A/G66A, F171A/T172A, N279A/F280A, F280A/R281A) but was not altered after Ala substitution of two non-adjacent AAs within the same NBS (F171A/N177A). SDS-PAGE in the Cy5 cell surface-labeled form demonstrated that the mutants appeared at the cell surface in oocytes. Thus, groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the hP2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor.

Compartmentalization of the BCR in membrane rafts is important for its signaling capacity. Swiprosin-1/EFhd2 (Swip-1) is an EF-hand and coiled-coil–containing adaptor protein with predicted Src homology 3 (SH3) binding sites that we identified in membrane rafts. We showed previously that Swip-1 amplifies BCR-induced apoptosis; however, the mechanism of this amplification was unknown. To address this question, we overexpressed Swip-1 and found that Swip-1 amplified the BCR-induced calcium flux in WEHI231, B62.1, and Bal17 cells. Conversely, the BCR-elicited calcium flux was strongly attenuated in Swip-1–silenced WEHI231 cells, and this was due to a decreased calcium mobilization from intracellular stores. Complementation of Swip-1 expression in Swip-1–silenced WEHI231 cells restored the BCR-induced calcium flux and enhanced spleen tyrosine kinase (Syk) tyrosine phosphorylation and activity as well as SLP65/BLNK/BASH and phospholipase C γ2 (PLCγ2) tyrosine phosphorylation. Furthermore, Swip-1 induced the constitutive association of the BCR itself, Syk, and PLCγ2 with membrane rafts. Concomitantly, Swip-1 stabilized the association of BCR with tyrosine-phosphorylated proteins, specifically Syk and PLCγ2, and enhanced the constitutive interaction of Syk and PLCγ2 with Lyn. Interestingly, Swip-1 bound to the rSH3 domains of the Src kinases Lyn and Fgr, as well as to that of PLCγ. Deletion of the predicted SH3-binding region in Swip-1 diminished its association and that of Syk and PLCγ2 with membrane rafts, reduced its interaction with the SH3 domain of PLCγ, and diminished the BCR-induced calcium flux. Hence, Swip-1 provides a membrane scaffold that is required for the Syk-, SLP-65–, and PLCγ2-dependent BCR-induced calcium flux.

Extracellular purines have multiple functional roles in development, plastic remodelling, and regeneration of the CNS by stimulating certain P2X/Y receptor (R) subtypes. In the present study we elucidated the involvement of P2YRs in neuronal fibre outgrowth in the developing nervous system. We particularly focused on the P2Y1R subtype and the dopaminergic system, respectively. For this purpose, we used organotypic slice co-cultures consisting of the ventral tegmental area/substantia nigra (VTA/SN) and the prefrontal cortex (PFC). After detecting the presence of the P2Y1R in VTA/SN, PFC, and on outgrowing fibres in the border region (e.g. on glial processes) connecting both brain slices, we could show that pharmacological modulation of the receptor influenced neuronal fibre outgrowth. Biocytin-tracing and tyrosine hydroxylase-immunolabelling together with quantitative image analysis revealed a significant increase in fibre growth in the border region of the co-cultures after treatment with ADPβS (P2Y1,12,13R agonist). The observed stimulatory potential of ADPβS was inhibited by pre-treatment with the P2X/YR antagonist PPADS. In P2Y1R knockout (P2Y1R(-/-)) mice, the ADPβS-induced stimulatory effect was absent, while growth was significantly enhanced in the co-cultures of the respective wild-type. This observation was confirmed in entorhino-hippocampal co-cultures, an example of a different projection system, expressing the P2Y1R. Using wortmannin and PD98059 we further showed that PI3K/Akt and MAPK/ERK cascades are involved in the mechanism underlying ADPβS-induced fibre growth. In conclusion, the data of this study clearly indicate that activation of the P2Y1R stimulates fibre growth and thereby emphasises the general role of this particular receptor subtype during development and regeneration.

Background and Purpose It is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane. Experimental Approach A molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP. Key Results A series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,β-methylene ATP (α,β-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,β-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression. Conclusions and Implications In conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.

Neurogenesis requires the balance between the proliferation of newly formed progenitor cells and subsequent death of surplus cells. RT-PCR and immunocytochemistry demonstrated the presence of P2X7 receptor mRNA and immunoreactivity in cultured neural progenitor cells (NPCs) prepared from the adult mouse subventricular zone (SVZ). Whole-cell patch-clamp recordings showed a marked potentiation of the inward current responses both to ATP and the prototypic P2X7 receptor agonist dibenzoyl-ATP (Bz-ATP) at low Ca(2+) and zero Mg(2+) concentrations in the bath medium. The Bz-ATP-induced currents reversed their polarity near 0 mV; in NPCs prepared from P2X7(-/-) mice, Bz-ATP failed to elicit membrane currents. The general P2X/P2Y receptor antagonist PPADS and the P2X7 selective antagonists Brilliant Blue G and A-438079 strongly depressed the effect of Bz-ATP. Long-lasting application of Bz-ATP induced an initial current, which slowly increased to a steady-state response. In combination with the determination of YO-PRO uptake, these experiments suggest the dilation of a receptor-channel and/or the recruitment of a dye-uptake pathway. Ca(2+)-imaging by means of Fura-2 revealed that in a Mg(2+)-deficient bath medium Bz-ATP causes [Ca(2+)](i) transients fully depending on the presence of external Ca(2+). The MTT test indicated a concentration-dependent decrease in cell viability by Bz-ATP treatment. Correspondingly, Bz-ATP led to an increase in active caspase 3 immunoreactivity, indicating a P2X7-controlled apoptosis. In acute SVZ brain slices of transgenic Tg(nestin/EGFP) mice, patch-clamp recordings identified P2X7 receptors at NPCs with pharmacological properties identical to those of their cultured counterparts. We suggest that the apoptotic/necrotic P2X7 receptors at NPCs may be of particular relevance during pathological conditions which lead to increased ATP release and thus could counterbalance the ensuing excessive cell proliferation.

The aim of the present experiments was to clarify the subunit stoichiometry of P2X2/3 and P2X2/6 receptors, where the same subunit (P2X2) forms a receptor with two different partners (P2X3 or P2X6). For this purpose, four non-functional Ala mutants of the P2X2, P2X3, and P2X6 subunits were generated by replacing single, homologous amino acids particularly important for agonist binding. Co-expression of these mutants in HEK293 cells to yield the P2X2 WT/P2X3 mutant or P2X2 mutant/P2X3 WT receptors resulted in a selective blockade of agonist responses in the former combination only. In contrast, of the P2X2 WT/P2X6 mutant and P2X2 mutant/P2X6 WT receptors, only the latter combination failed to respond to agonists. The effects of α,β-methylene-ATP and 2-methylthio-ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca(2+) transients by the Ca(2+)-imaging method. Protein labeling, purification, and PAGE confirmed the assembly and surface trafficking of the investigated WT and WT/mutant combinations in Xenopus laevis oocytes. In conclusion, both electrophysiological and biochemical investigations uniformly indicate that one subunit of P2X2 and two subunits of P2X3 form P2X2/3 heteromeric receptors, whereas two subunits of P2X2 and one subunit of P2X6 constitute P2X2/6 receptors. Further, it was shown that already two binding sites of the three possible ones are sufficient to allow these receptors to react with their agonists.

Astrocytes express purinergic receptors that are involved in glial–neuronal cell communication. Experiments were conducted to characterize the expression of functional P2X/P2Y nucleotide receptors in glial cells of mixed cortical cell cultures of the rat. The vast majority of these cells was immunopositive for glial fibrillary acidic protein (GFAP) and was considered therefore astrocyte-like; for the sake of simplicity they were termed “astroglia” throughout. Astroglia expressed predominantly P2X 4,6,7 as well as P2Y 1,2 receptor-subtypes. Less intensive immunostaining was also found for P2X 5 and P2Y 4,6,13,14 receptors. Pressure application of ATP and a range of agonists selective for certain P2X or P2Y receptor-subtypes caused a concentration-dependent increase of intracellular Ca 2+ ([Ca 2+ ] i ). Of the agonists tested, only the P2X 1,3 receptor-selective α,β-methylene ATP was ineffective. Experiments with Ca 2+ -free solution and cyclopiazonic acid, an inhibitor of the endoplasmic Ca 2+ -ATPase, indicated that the [Ca 2+ ] i response to most nucleotides, except for ATP and 2′,3′- O -(benzoyl-4-benzoyl)-ATP, was due primarily to the release of Ca 2+ from intracellular stores. A Gprotein–mediated release of Ca 2+ is the typical signaling mechanism of various P2Y receptor-subtypes, whose presence was confirmed also by cross-desensitization experiments and by using selective antagonists. Thus, our results provide direct evidence that astroglia in mixed cortical cell cultures express functional P2Y (P2Y 1,2,6,14 and probably also P2Y 4 ) receptors. Several unidentified P2X receptors, including P2X 7 , may also be present, although they appear to only moderately participate in the regulation of [Ca 2+ ] i . The rise of [Ca 2+ ] i is due in this case to the transmembrane flux of Ca 2+ via the P2X receptor-channel. In conclusion, P2Y rather than P2X receptor-subtypes are involved in modulating [Ca 2+ ] i of cultured astroglia and thereby may play an important role in cell-to-cell signaling.

Carma-1 is required for B cell receptor-/CD40- and T cell receptor-/CD28-induced B- and T-cell activation via JNK and NF-betaB. In B cells, Carma-1 becomes phosphorylated by PKCbeta, leading to its oligomerization. Subsequent Bcl10 binding induces IKKbeta-activation and, thereby, canonical NF-KB signalling. Despite these findings it is still unknown how exactly Carma-1 is connected to the plasma membrane and to the IKK-complex. Therefore, we purified Carma-1 complexes from mouse CH12 B cells using anti-Carma-1 affinity columns. Mass spectrometric analyses of the column eluates demonstrated the presence of Carma-1 as well as three previously uncharacterized adaptor proteins in B cells, one of which was the Trk-fused gene (Tfg), an adaptor protein containing PB1 and coiledcoil domains. Whereas Tfg was originally identified as fusion partner of oncogenic Trk tyrosine kinase mutants, the normal cellular homologue of Tfg has so far not been described in B cells. However, Tfg has been shown in other systems to interact with IKKgamma and to enhance TNFinduced NF-KB activation. Tfg and Carma-1 co-localized at the plasma membrane and perinuclear structures in B cells. We further corroborated the interactions of Tfg, IKKgamma and Carma-1 by Blue Native gel electrophoresis, where Carma-1 and Tfg formed a 0.7–1 MDa complex. Ectopic expression of Tfg increased the molecular mass of IKKgamma complexes, fused IKKgamma, Bcl10 and Carma-1 complexes to a ~2 MDa complex, and increased basal and CD40-induced canonical activity of NF-KB and IKKbeta. In contrast, shRNA-mediated silencing of Tfg decreased CD40-induced IKKbeta activity. Very interestingly, in primary B cells, highest expression of Tfg was detected in marginal zone and B1 B cells, and Carma-1 and Tfg formed complexes in these B cells. Since Carma-1 is required for marginal zone B cell and B1 B cell development, we suggest that a functional interaction between Carma-1 and Tfg contributes to development and maintenance of these cells by means of canonical NF-KB signals.