Nationwide quality assurance of high-throughput diagnostic molecular testing during the SARS-CoV-2 pandemic: role of the Belgian National Reference Centre.

Since the onset of the coronavirus disease (COVID-19) pandemic in Belgium, UZ/KU Leuven has played a crucial role as the National Reference Centre (NRC) for respiratory pathogens, to be the first Belgian laboratory to develop and implement laboratory developed diagnostic assays for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and later to assess the quality of commercial kits. To meet the growing demand for decentralised testing, both clinical laboratories and government-supported high-throughput platforms were gradually deployed across Belgium. Consequently, the role of the NRC transitioned from a specialised testing laboratory to strengthening capacity and coordinating quality assurance. Here, we outline the measures taken by the NRC, the national public health institute Sciensano and the executing clinical laboratories to ensure effective quality management of molecular testing throughout the initial two years of the pandemic (March 2020 to March 2022).

Evaluating Diagnostic Accuracy of Saliva Sampling Methods for Severe Acute Respiratory Syndrome Coronavirus 2 Reveals Differential Sensitivity and Association with Viral Load.

Nasopharyngeal swabs are considered the preferential collection method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnostics. Less invasive and simpler alternative sampling procedures, such as saliva collection, are desirable. We compared saliva specimens and nasopharyngeal (NP) swabs with respect to sensitivity in detecting SARS-CoV-2. A nasopharyngeal and two saliva specimens (collected by spitting or oral swabbing) were obtained from >2500 individuals. All samples were tested by RT-qPCR, detecting RNA of SARS-CoV-2. The test sensitivity was compared on the two saliva collections with the nasopharyngeal specimen for all subjects and stratified by symptom status and viral load. Of the 2850 patients for whom all three samples were available, 105 were positive on NP swab, whereas 32 and 23 were also positive on saliva spitting and saliva swabbing samples, respectively. The sensitivity of the RT-qPCR to detect SARS-CoV-2 among NP-positive patients was 30.5% (95% CI, 1.9%-40.2%) for saliva spitting and 21.9% (95% CI, 14.4%-31.0%) for saliva swabbing. However, when focusing on subjects with medium to high viral load, sensitivity on saliva increased substantially: 93.9% (95% CI, 79.8%-99.3%) and 76.9% (95% CI, 56.4%-91.0%) for spitting and swabbing, respectively, regardless of symptomatic status. Our results suggest that saliva cannot readily replace nasopharyngeal sampling for SARS-CoV-2 diagnostics but may enable identification of the most contagious cases with medium to high viral loads.

Repurposing HAMI3379 to Block GPR17 and Promote Rodent and Human Oligodendrocyte Differentiation.

Identification of additional uses for existing drugs is a hot topic in drug discovery and a viable alternative to de novo drug development. HAMI3379 is known as an antagonist of the cysteinyl-leukotriene CysLT2 receptor, and was initially developed to treat cardiovascular and inflammatory disorders. In our study we identified HAMI3379 as an antagonist of the orphan G protein-coupled receptor GPR17. HAMI3379 inhibits signaling of recombinant human, rat, and mouse GPR17 across various cellular backgrounds, and of endogenous GPR17 in primary rodent oligodendrocytes. GPR17 blockade by HAMI3379 enhanced maturation of primary rat and mouse oligodendrocytes, but was without effect in oligodendrocytes from GPR17 knockout mice. In human oligodendrocytes prepared from inducible pluripotent stem cells, GPR17 is expressed and its activation impaired oligodendrocyte differentiation. HAMI3379, conversely, efficiently favored human oligodendrocyte differentiation. We propose that HAMI3379 holds promise for pharmacological exploitation of orphan GPR17 to enhance regenerative strategies for the promotion of remyelination in patients.

5-HT7 Receptor Antagonists with an Unprecedented Selectivity Profile.

Selective leads: In this study, we generated a new series of serotonin 5-HT7 receptor antagonists. Their synthesis, structure-activity relationships, and selectivity profiles are reported. This series includes 5-HT7 antagonists with unprecedented high selectivity for the 5-HT7 receptor, setting the stage for lead optimization of drugs acting on a range of neurological targets.

The tau positron-emission tomography tracer AV-1451 binds with similar affinities to tau fibrils and monoamine oxidases.

BACKGROUND: Lilly/Avid's AV-1451 is one of the most advanced tau PET tracers in the clinic. Although results obtained in Alzheimer's disease patients are compelling, discrimination of tracer uptake in healthy individuals and patients with supranuclear palsy (PSP) is less clear as there is substantial overlap of signal in multiple brain regions. Moreover, accurate quantification of [18 F]AV-1451 uptake in Alzheimer's disease may not be possible. OBJECTIVES: The aim of the present study was to characterize the in vitro binding of AV-1451 to understand and identify potential off-target binding that could explain the poor discrimination observed in PSP patients. METHODS: [3 H]AV-1451 and AV-1451 were characterized in in vitro binding assays using recombinant and native proteins/tissues from postmortem samples of controls and Alzheimer's disease and PSP patients. RESULTS: [3 H]AV-1451 binds to multiple sites with nanomolar affinities in brain homogenates and to tau fibrils isolated from Alzheimer's disease or PSP patients. [3 H]AV-1451 also binds with similarly high affinities in brain homogenates devoid of tau pathology. This unexpected binding was demonstrated to be because of nanomolar affinities of [3 H]AV-1451 for monoamine oxidase A and B enzymes. CONCLUSIONS: High affinity of AV-1451 for monoamine oxidase proteins may limit its utility as a tau PET tracer in PSP and Alzheimer's disease because of high levels of monoamine oxidase expression in brain regions also affected by tau deposition, especially if monoamine oxidase levels change over time or with a treatment intervention. © 2017 International Parkinson and Movement Disorder Society.

The Orphan Receptor GPR17 Is Unresponsive to Uracil Nucleotides and Cysteinyl Leukotrienes.

Pairing orphan G protein­coupled receptors (GPCRs) with their cognate endogenous ligands is expected to have a major impact on our understanding of GPCR biology. It follows that the reproducibility of orphan receptor ligand pairs should be of fundamental importance to guide meaningful investigations into the pharmacology and function of individual receptors. GPR17 is an orphan receptor characterized by some as a dualistic uracil nucleotide/cysteinyl leukotriene receptor and by others as inactive toward these stimuli altogether. Whereas regulation of central nervous system myelination by GPR17 is well established, verification of activity of its putative endogenous ligands has proven elusive so far. Herein we report that uracil nucleotides and cysteinyl leukotrienes do not activate human, mouse, or rat GPR17 in various cellular backgrounds, including primary cells, using eight distinct functional assay platforms based on labelfree pathway-unbiased biosensor technologies, as well as canonical second-messenger or biochemical assays. Appraisal of GPR17 activity can neither be accomplished with co-application of both ligand classes, nor with exogenous transfection of partner receptors (nucleotide P2Y12, cysteinyl-leukotriene CysLT1) to reconstitute the elusive pharmacology. Moreover, our study does not support the inhibition of GPR17 by the marketed antiplatelet drugs cangrelor and ticagrelor, previously suggested to antagonize GPR17. Whereas our data do not disagree with a role of GPR17 per se as an orchestrator of central nervous system functions, they challenge the utility of the proposed (ant)agonists as tools to imply direct contribution of GPR17 in complex biologic settings.

Decoding signaling and function of the orphan G protein-coupled receptor GPR17 with a small-molecule agonist.

Replacement of the lost myelin sheath is a therapeutic goal for treating demyelinating diseases of the central nervous system (CNS), such as multiple sclerosis (MS). The G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) GPR17, which is phylogenetically closely related to receptors of the "purinergic cluster," has emerged as a modulator of CNS myelination. However, whether GPR17-mediated signaling positively or negatively regulates this critical process is unresolved. We identified a small-molecule agonist, MDL29,951, that selectively activated GPR17 even in a complex environment of endogenous purinergic receptors in primary oligodendrocytes. MDL29,951-stimulated GPR17 engaged the entire set of intracellular adaptor proteins for GPCRs: G proteins of the Gα(i), Gα(s), and Gα(q) subfamily, as well as ß-arrestins. This was visualized as alterations in the concentrations of cyclic adenosine monophosphate and inositol phosphate, increased Ca²âº flux, phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), as well as multifeatured cell activation recorded with label-free dynamic mass redistribution and impedance biosensors. MDL29,951 inhibited the maturation of primary oligodendrocytes from heterozygous but not GPR17 knockout mice in culture, as well as in cerebellar slices from 4-day-old wild-type mice. Because GPCRs are attractive targets for therapeutic intervention, inhibiting GPR17 emerges as therapeutic strategy to relieve the oligodendrocyte maturation block and promote myelin repair in MS.

Loss of metabotropic glutamate receptor-mediated regulation of glutamate transport in chemically activated astrocytes in a rat model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurones accompanied by intense gliosis in lesioned areas of the brain and spinal cord. Glutamate-mediated excitotoxicity resulting from impaired astroglial uptake constitutes one of the current pathophysiological hypotheses explaining the progression of the disease. In this study, we examined the regulation of glutamate transporters by type 5 metabotropic glutamate receptor (mGluR5) in activated astrocytes derived from transgenic rats carrying an ALS-related mutated human superoxide dismutase 1 (hSOD1(G93A)) transgene. Cells from transgenic animals and wild-type littermates showed similar expression of glutamate-aspartate transporter and glutamate transporter 1 (GLT-1) after in vitro activation, whereas cells carrying the hSOD1 mutation showed a three-fold higher expression of functional mGluR5, as observed in the spinal cord of end-stage animals. In cells from wild-type animals, (S)-3,5-dihydroxyphenylglycine (DHPG) caused an immediate protein kinase C (PKC)-dependent up-regulation of aspartate uptake that reflected the activation of GLT-1. Although this effect was mimicked in both cultures by direct activation of PKC using phorbol myristate acetate, DHPG failed to up-regulate aspartate uptake in cells derived from the transgenic rats. The failure of activated mGluR5 to increase glutamate uptake in astrocytes derived from this animal model of ALS supports the theory of glutamate excitotoxicity in the pathogenesis of the disease.

Acute up-regulation of glutamate uptake mediated by mGluR5a in reactive astrocytes.

Excitatory transmission in the CNS necessitates the existence of dynamic controls of the glutamate uptake achieved by astrocytes, both in physiological conditions and under pathological circumstances characterized by gliosis. In this context, this study was aimed at evaluating the involvement of group I metabotropic glutamate receptors (mGluR) in the regulation of glutamate transport in a model of rat astrocytes undergoing in vitro activation using a cocktail of growth factors (G5 supplement). The vast majority of the cells were found to take up aspartate, mainly through the glutamate/aspartate transporter (GLAST), and at least 60% expressed functional mGluR5a. When exposed for 15 s to the selective group I mGluR agonist (S)-3,5-dihydroxyphenylglycine, reactive astrocytes showed a significant increase in their capacity to take up aspartate. This effect was confirmed at the single-cell level, since activation of mGluRs significantly increased the initial slope of aspartate-dependent Na+ entry associated with the activity of glutamate transporters. This up-regulation was inhibited by an antagonist of mGluR5 and, more importantly, was sensitive to a specific glutamate transporter 1 (GLT-1) blocker. The acute influence of mGluR5 on aspartate uptake was phospholipase C- and protein kinase C-dependent, and was mimicked by phorbol esters. We conclude that mGluR5a contributes to a dynamic control of GLT-1 function in activated astrocytes, acting as a glial sensor of the extracellular glutamate concentration in order to acutely regulate the excitatory transmission.

Molecular and functional characterisation of glutamate transporters in rat cortical astrocytes exposed to a defined combination of growth factors during in vitro differentiation.

In vitro culture of astroglial progenitors can be obtained from early post-natal brain tissues and several methods have been reported for promoting their maturation into differentiated astrocytes. Hence, a combination of several nutriments/growth factors -- the G5 supplement (insulin, transferrin, selenite, biotin, hydrocortisone, fibroblast growth factor and epidermal growth factor) -- is widely used as a culture additive favouring the growth, differentiation and maturation of primary cultured astrocytes. Considering the key role played by glial cells in the clearance of glutamate in the synapses, cultured astrocytes are frequently used as a model for the study of glutamate transporters. Indeed, it has been shown that when tested separately, growth factors influence the expression and activity of the GLAST and GLT-1. The present study aimed at characterising the functional expression of these transporters during the time course of differentiation of cultured cortical astrocytes exposed to the supplement G5. After a few days, the vast majority of cells exposed to this supplement adopted a typical stellate morphology (fibrous or type II astrocytes) and showed intense expression of the glial fibrillary acidic protein. Both RT-PCR and immunoblotting studies revealed that the expression of both GLAST and GLT-1 rapidly increased in these cells. While this was correlated with a significant increase in specific uptake of radiolabelled aspartate, fluorescence monitoring of the Na+ influx associated with glutamate transporters activity revealed that the exposure to the G5 supplement considerably increased the percentage of cells participating in the uptake. Biochemical and pharmacological studies revealed that this activity did not involve GLT-1 but most likely reflected an increase in GLAST-mediated uptake. Together, these data indicate that the addition of this classical combination of growth factors and nutriments drives the rapid differentiation toward a homogenous culture of fibrous astrocytes expressing functional glutamate transporters.

In vitro differentiated neural stem cells express functional glial glutamate transporters.

The possibility to isolate stem cells from the adult central nervous system and to maintain and propagate these cells in vitro has raised a general interest with regards to their use in cell replacement therapy for degenerative brain diseases. Considering the critical role played by astrocytes in the control of glutamate homeostasis, we have characterised the expression of functional glutamate transporters in neural stem cells exposed to selected culture conditions favouring their differentiation into astrocytes. Commonly, neural stem cells proliferate in suspension as neurospheres in serum-free medium. The addition of serum or a supplement of growth factors (G5) to the culture medium was found to trigger cell adhesion on coated surfaces and to favour their differentiation. Indeed, after 7 days in these conditions, the vast majority of the cells adopted markedly distinct morphologies corresponding to protoplasmic (with serum) or fibrous (with G5 supplement) astrocytes and approximately 35-40% acquired the expression of the glial fibrillary acidic protein (GFAP). Immunocytochemical analysis also revealed that the treatments with serum or with the G5 supplement triggered the expression of the glial glutamate transporters GLT-1 (35 and 21%, respectively) and GLAST (29 and 69%, respectively). This effect was correlated with a robust increase in the Na+ -dependent [3H]-d-aspartate uptake, which was partially inhibited by dihydrokainate, a selective blocker of GLT-1. Together, these results indicate that in vitro differentiation of cultured neural stem cells can give rise to distinct populations of astrocytes expressing functional glutamate transporters.

Induction of glial glutamate transporters in adult mesenchymal stem cells.

Adult bone marrow mesenchymal stem cells are multipotent cells that can differentiate into a variety of mesodermal tissues. Recent studies have reported on their ability to also evolve into non-mesodermal cells, especially neural cells. While most of these studies revealed that manipulating these cells triggers the expression of typical neurone markers, less is known about the induction of neuronal- or glial-related physiological properties. The present study focused on the characterisation of glutamate transporters expression and activity in rat mesenchymal stem cells grown in culture conditions favouring their differentiation into astroglial cells. Ten days exposure of the cells to the culture supplement G5 was found to increase the expression of nestin (neuro-epithelial stem cell intermediate filament), an intermediate filament protein expressed by neural stem cells. Simultaneously, a robust induction of the high-affinity glutamate transporter GLT-1 (and GLAST) expression was detected by RT-PCR and immunocytochemistry. This expression was correlated with a highly significant increase in the Na+-dependent [3H]D-aspartate uptake. Finally, while glial fibrillary acidic protein immunoreactivity could not be detected, the induced expression of the astrocytic enzyme glutamine synthetase was demonstrated. These results indicate that in vitro differentiation of adult mesenchymal stem cells in neural precursors coincides with the induction of functional glutamate transport systems. Although the astrocytic nature of these cells remains to be confirmed, this observation gives support to the study of mesenchymal stem cells as a promising tool for the treatment of neurological diseases involving glutamate excitoxicity.

IS231-MIC231 elements from Bacillus cereus sensu lato are modular.

Summary IS231A was originally discovered in Bacillus thuringiensis as a typical 1.6 kb insertion sequence (IS) displaying 20 bp inverted repeats (IR) flanking a transposase gene. A first major variation of this canonical organization was found in MIC231A1. This mobile insertion cassette (MIC), delineated by IS231A-related extremities, contained an active d-stereospecific endopeptidase (adp) gene instead of a transposase. Interestingly, it was shown that MIC231A1 can be mobilized in trans by the IS231A transposase. In this paper, we show that this family of IS231-MIC231 elements can be extended to a broad range of related entities displaying higher levels of structural complexity. Several IS231A-like elements contained, upstream of their transposase gene, passenger genes coding for putative antibiotic resistances or regulatory factors. Furthermore, the diversity of the MIC231 elements ranged from empty cassettes to structures carrying up to three passenger genes. Among these, MIC231V carried, in addition to an adp gene, an active fosfomycin resistance determinant. In vivo transposition assays showed that MIC231V is also trans-activated by the IS231A transposase. These results lend further support to the potential contribution of these modular mobile elements to the genome plasticity of the Bacillus cereus/B. thuringiensis group.