In vitro study of uptake and synthesis of creatine and its precursors by cerebellar granule cells and astrocytes suggests some hypotheses on the physiopathology of the inherited disorders of creatine metabolism

<p>Abstract</p> <p>Background</p> <p>The discovery of the inherited disorders of creatine (Cr) synthesis and transport in the last few years disclosed the importance of blood Cr supply for the normal functioning of the brain. These putatively rare diseases share a common pathogenetic mechanism (the depletion of brain Cr) and similar phenotypes characterized by mental retardation, language disturbances, seizures and movement disorders. In the effort to improve our knowledge on the mechanisms regulating Cr pool inside the nervous tissue, Cr transport and synthesis and related gene transcripts were explored in primary cultures of rat cerebellar granule cells and astrocytes.</p> <p>Methods</p> <p>Cr uptake and synthesis were explored in vitro by incubating monotypic primary cultures of rat type I astrocytes and cerebellar granule cells with: a) D₃-Creatine (D₃Cr) and D3Cr plus β-guanidinopropionate (GPA, an inhibitor of Cr transporter), and b) labelled precursors of Guanidinoacetate (GAA) and Cr (Arginine, Arg; Glycine, Gly). Intracellular D3Cr and labelled GAA and Cr were assessed by ESI-MS/MS. Creatine transporter (<it>CT1</it>), L-arginine:glycine amidinotransferase (<it>AGAT</it>), and S-adenosylmethionine:guanidinoacetate N-methyltransferase (<it>GAMT</it>) gene expression was assessed in the same cells by real time PCR.</p> <p>Results</p> <p>D3Cr signal was extremely high in cells incubated with this isotope (labelled/unlabelled Cr ratio reached about 10 and 122, respectively in cerebellar granule cells and astrocytes) and was reduced by GPA. Labelled Arg and Gly were taken up by the cells and incorporated in GAA, whose concentration paralleled that of these precursors both in the extracellular medium and inside the cells (astrocytes). In contrast, the increase of labelled Cr was relatively much more limited since labelled Cr after precursors' supplementation did not exceed 2,7% (cerebellar granule cells) and 21% (astrocytes) of unlabelled Cr. Finally, <it>AGAT, GAMT </it>and <it>SLC6A8 </it>were expressed in both kind of cells.</p> <p>Conclusions</p> <p>Our results confirm that both neurons and astrocytes have the capability to synthesize and uptake Cr, and suggest that at least in vitro intracellular Cr can increase to a much greater extent through uptake than through <it>de novo </it>synthesis. Our results are compatible with the clinical observations that when the Cr transporter is defective, intracellular Cr is absent despite the brain should be able to synthesize it. Further research is needed to fully understand to what extent our results reflect the in vivo situation.</p

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Concomitant Onychomadesis and Skin Rash in a 3-Year-Old Child Affected by Hand, Foot, and Mouth Disease

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Shigella modulation of polyamines during the invasion of host cells

Polyamines are small molecules found in all cells and associated with a wide variety of physiological processes. In bacterial pathogens the modulation of polyamine content could represent a strategy to optimize bacterial fitness within the host. Shigella, the etiological agent of bacillary dysentery, in contrast to its innocuous ancestor Escherichia coli, has a polyamine profile characterized by high level of intracellular spermidine. To understand the role of polyamines in the Shigella-host interaction, we analysed the expression of host cell genes involved in the biogenesis and back-conversion of polyamines during Shigella infection. Preliminary data indicate that the acetyl polyamine oxidase (APAO) is induced at early stages of Shigella infection, while induction of the spermine/spermidine acetyltransferase and of the spermine oxidase is detected later during the infection. These observations lead us to speculate that the early release of H2O2 as secondary product of APAO activity might represent a signal for Shigella to activate the expression of mdtJI efflux pump operon in order to export bacterial polyamines which have a scavenger function during oxidative stress conditions

Expression profile of efflux pumps during the intracellular life of Shigella

In several bacterial pathogens, efflux pumps, besides exporting antimicrobial agents, play a role in bacterial pathogenicity. Shigella, the causative agent of bacillary dysentery, shares a high homology with its commensal ancestor Escherichia coli but, in contrast to E. coli, Shigella is submitted to intense gene decay and has lost many E. coli metabolic and housekeeping functions. By genome analysis we observed that 14, out of 20, operons encoding efflux pumps systems have been conserved in Shigella. To understand the potential role of efflux pumps in Shigella pathogenicity, we monitored their expression during invasion of macrophages and epithelial cells. The data obtained indicate that i) EmrKY efflux pump is notably and specifically induced within macrophages and its loss negatively affects Shigella’s fitness; ii) AcrAB, the main efflux pump in E. coli, is downregulated during intracellular life. Our observations reveal also that, during infection, Shigella modulates expression of specific efflux pumps. Experiments are in progress to investigate the role of these efflux pumps and the environmental stimuli that affect their expression