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3,4‐Methylenedioxymethamphetamine (MDMA, 'ecstasy') is re‐emerging in clinical settings as a candidate for the treatment of specific neuropsychiatric disorders (e.g. post‐traumatic stress disorder) in combination with psychotherapy. MDMA is a psychoactive drug, typically regarded as an empathogen or entactogen, which leads to transporter‐mediated monoamine release. Despite its therapeutic potential, MDMA can induce dose‐, individual‐, and context‐dependent untoward effects outside safe settings. In this study, we investigated whether three new methylenedioxy bioisosteres of MDMA improve its off‐target profile. In vitro methods included radiotracer assays, transporter electrophysiology, bioluminescence resonance energy transfer and fluorescence‐based assays, pooled human liver microsome/S9 fraction incubations, metabolic stability studies, isozyme mapping, and liquid chromatography coupled to high‐resolution mass spectrometry. In silico methods included molecular docking. Compared with MDMA, all three MDMA bioisosteres (ODMA, TDMA, and SeDMA) showed similar pharmacological activity at human serotonin, dopamine, and norepinephrine transporters (hSERT, hDAT, and hNET, respectively) but decreased agonist activity at 5‐HT2A/2B/2C receptors. Regarding their hepatic metabolism, they differed from MDMA, with N‐demethylation being the only metabolic route shared, and without forming phase II metabolites. In addition, TDMA showed an enhanced intrinsic clearance in comparison to its congeners. Additional screening for their interaction with human organic cation transporters (hOCTs) and plasma membrane monoamine transporter (hPMAT) revealed a weaker interaction of the MDMA analogs with hOCT1, hOCT2, and hPMAT. Our findings suggest that these new MDMA bioisosteres might constitute appealing therapeutic alternatives to MDMA, sparing the primary pharmacological activity at hSERT, hDAT, and hNET, but displaying a reduced activity at 5‐HT2A/2B/2C receptors and alternative hepatic metabolism. Whether these MDMA bioisosteres may pose lower risk alternatives to the clinically re‐emerging MDMA warrants further studies. [ABSTRACT FROM AUTHOR]

This article discusses the phenomenon of pseudothrombocytopenia (PTP), which refers to falsely low platelet counts in blood samples due to platelet agglutination during sample storage. The study compared the accuracy and stability of platelet counts in PTP subjects using different anticoagulants and storage times. The results showed that K3EDTA was the most reliable anticoagulant for immediate platelet counting, while alternative anticoagulants were better at stabilizing platelet counts over time. However, all anticoagulants were unable to prevent a time-dependent decrease in platelet count in some samples, leading to thrombocytopenia. The study suggests that microscopic examination of blood samples is still necessary to confirm platelet counts in PTP subjects. [Extracted from the article]

Summary: Abnormalities of platelet function were reported in patients with severe COVID‐19 (severe‐C), but few data are available in patients with mild COVID‐19 (mild‐C) and after COVID‐19 recovery. The aim of this study was to investigate platelet parameters in mild‐C patients (n = 51), with no evidence of pneumonia, and severe‐C patients (n = 49), during the acute phase and after recovery, compared to 43 healthy controls. Both mild‐C and severe‐C patients displayed increased circulating activated platelets, low δ‐granule content (ADP, serotonin), impaired platelet activation by collagen (light transmission aggregometry) and impaired platelet thrombus formation on collagen‐coated surfaces under controlled flow conditions (300/s shear rate). The observed abnormalities were more marked in severe‐C patients than in mild‐C patients. Overall, 61% (30/49) of mild‐C and 73% (33/45) of severe‐C patients displayed at least one abnormal platelet parameter. In a subgroup of just 13 patients who showed no persisting signs/symptoms of COVID‐19 and were re‐evaluated at least 1 month after recovery, 11 of the 13 subjects exhibited normalization of platelet parameters. In conclusion, mild abnormalities of platelet parameters were present not only in severe‐C but also, albeit to a lesser extent, in mild‐C patients during the acute phase of COVID‐19 and normalized in most tested patients after clinical recovery. [ABSTRACT FROM AUTHOR]

The highly homologous neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the Manduca sexta larva, are members of the [Na.sup.+]/[Cl.sup.-] dependent transporter family. Recent evidence indicates that transporters of this family form constitutive oligomers. CAATCH1 and KAAT1 give rise to specific kinds of current depending on the transported amino acid, cotransported ion, pH, and membrane voltage. Different substrates induce notably distinct transport-associated currents in the two proteins that represent useful tools in structural-functional studies. To determine whether KAAT1 and CAATCH1 form functional oligomers, we have constructed four concatameric proteins for electrophysiological analysis, consisting of one KAAT1 protein covalently linked to another KAAT1 (K-K concatamer) or to CAATCH1 (K-C concatamer) and vice versa (C-C concatamer and C-K concatamer), and eight constructs where the two transporters were linked to yellow or cyan fluorescent protein in the N[H.sub.2] or COOH terminus, to determine the oligomer formation and the relative distance between the different subunits by fluorescence resonance energy transfer (FRET) analysis. Heterologous expression of the concatenated constructs and coinjection of the original proteins in different proportions allowed us to compare the characteristics of the currents to those of the oocytes expressing only the wild-type proteins. All the constructs were fully active, and their electrophysiological behavior was consistent with the activity as monomeric proteins. However, the FRET studies indicate that these transporters form oligomers in agreement with the LeU[T.sub.Aa] atomic structure and confirm that the COOH termini of the adjacent subunits are closer than N[H.sub.2] termini. transport; oligomerization; Slc6; fret

The ability of the two highly homologous [Na.sup.+]/[Cl.sup.-]-dependent neutral amino acid transporters KAAT1 and CAATCH1, cloned from the midgut epithelium of the larva Manduca sexta, to transport different amino acids depends on the cotransported ion, on pH, and on the membrane voltage. Different organic substrates give rise to transport-associated currents with their own characteristics, which are notably distinct between the two proteins. Differences in amplitude, kinetics, and voltage dependence of the transport-associated currents have been observed, as well as different substrate selectivity patterns measured by radioactive amino acid uptake assays. These diversities represent useful tools to investigate the structural determinants involved in the substrate selectivity. To identify these regions, we built four chimeric proteins between the two transporters. These proteins, heterologously expressed in Xenopus laevis oocytes, were analyzed by two-electrode voltage clamp and uptake measurements. Initially, we exchanged the first three domains, obtaining the chimeras C3K9 and K3C9 (where numbers indicate the transmembrane domains and letters represent the original proteins), which showed electrophysiological and [[sup.3]H]amino acid uptake characteristics resembling those of KAAT1 and CAATCH1, respectively. Subsequent substitution of the last four domains in C3K9 and K3C9 gave the proteins C3K5C4 and K3C5K4, which showed the same behavior as KAAT1 and CAATCH1 in electrophysiological and transport determinations. These results suggest that in KAAT1 and CAATCH1, only the central transmembrane domains (from 4 to 8) of the protein are responsible for substrate selectivity. structure and function; Manduca sexta

KAAT1 is a neutral amino acid transporter activated by [K.sup.+] or by [Na.sup.+] (9). The protein shows significant homology with members of the [Na.sup.+]/[Cl.sup.-] -dependent neurotransmitter transporter super family. E59G KAAT1, expressed in Xenopus oocytes, exhibited a reduced leucine uptake [20-30% of wild-type (WT)], and kinetic analysis indicated that the loss of activity was due to reduction of [V.sub.max] and apparent affinity for substrates. Electrophysiological analysis revealed that E59G KAAT1 has presteady-state and uncoupled currents larger than WT but no leucine-induced currents. Site-directed mutagenesis analysis showed the requirement of a negative charge in position 59 of KAAT1. The analysis of permeant and impermeant methanethiosulfonate reagent effects confirmed the intracellular localization of glutamate 59. Because the 2-aminoethyl methanethiosulfonate hydrobromid inhibition was not prevented by the presence of [Na.sup.+] or leucine, we concluded that E59 is not directly involved in the binding of substrates. N-ethylmaleimide inhibition was qualitatively and quantitatively different in the two transporters, WT and E59G KAAT1, having the same cysteine residues. This indicates an altered accessibility of native cysteine residues due to a modified spatial organization of E59G KAAT1. The arginine modifier phenylglyoxal effect supports this hypothesis: not only cysteine but also arginine residues become more accessible to the modifying reagents in the mutant E59G. In conclusion, the results presented indicate that glutamate 59 plays a critical role in the three-dimensional organization of KAAT1. amino acid transport; structure/function; amino acid modifiers; Manduca sexta