30 research outputs found
Optical Coherence Tomography Imaging of Acute Stent Thrombosis After Stentys Stent Implantation
Cytotoxicity, metabolism, and isozyme mapping of the synthetic cannabinoids JWH-200, A-796260, and 5F-EMB-PINACA studied by means of in vitro systems
Intake of synthetic cannabinoids (SC), one of the largest classes of new psychoactive substances, was reported to be associated with acute liver damage but information about their hepatotoxic potential is limited. The current study aimed to analyze the hepatotoxicity including the metabolism-related impact of JWH-200, A-796260, and 5F-EMB-PINACA in HepG2 cells allowing a tentative assessment of different SC subclasses. A formerly adopted high-content screening assay (HCSA) was optimized using a fully automated epifluorescence microscope. Metabolism-mediated effects in the HCSA were additionally investigated using the broad CYP inhibitor 1-aminobenzotriazole. Furthermore, phase I metabolites and isozymes involved were identified by in vitro assays and liquid chromatographyāhigh-resolution tandem mass spectrometry. A strong cytotoxic potential was observed for the naphthoylindole SC JWH-200 and the tetramethylcyclopropanoylindole compound A-796260, whereas the indazole carboxamide SC 5F-EMB-PINACA showed moderate effects. Numerous metabolites, which can serve as analytical targets in urine screening procedures, were identified in pooled human liver microsomes. Most abundant metabolites of JWH-200 were formed by N-dealkylation, oxidative morpholine cleavage, and oxidative morpholine opening. In case of A-796260, most abundant metabolites included an oxidative morpholine cleavage, oxidative morpholine opening, hydroxylation, and dihydroxylation followed by dehydrogenation. Most abundant 5F-EMB-PINACA metabolites were generated by ester hydrolysis plus additional steps such as oxidative defluorination and hydroxylation. To conclude, the data showed that a hepatotoxicity of the investigated SC cannot be excluded, that metabolism seems to play a minor role in the observed effects, and that the extensive phase I metabolism is mediated by several isozymes making interaction unlikely
Serial Morphological and Functional Assessment of Drug-Eluting Balloon for In-Stent Restenotic Lesions Mechanisms of Action Evaluated With Angiography, Optical Coherence Tomography, and Fractional Flow Reserve
ObjectivesThis study sought to elucidate the underlying mechanism through which drug-eluting balloons (DEB) restore coronary blood flow, by assessing the coronary vessel before, immediately after, and at 6-month follow-up with angiography, optical coherence tomography (OCT), and fractional flow reserve (FFR).BackgroundIn-stent restenosis (ISR) treatment remains challenging. Drug-eluting balloons have been shown to be a valid treatment option in several studies. These studies focused on efficiency of the device, whereas the mechanisms of action of DEB in ISR treatment have not been investigated.MethodsIn this prospective, single-center observational study, patients with ISR were treated with a second-generation DEB. Serial angiographic, OCT, and FFR measurements were performed before and after the procedure, as well as at 6-month follow-up.ResultsTwenty-five patients were assigned to DEB treatment, with an angiographic and device success of 100% and 92%, respectively. Late luminal loss was 0.01 Ā± 0.43 mm. Median percent changes [interquartile range] between pre-and post-procedure, and post-procedure and follow-up were, respectively: lumen volume 75.1% increase [43.7 to 115.0], and 8% increase [ā14.0 to 25.8]; stent volume 23.7% increase [15.5 to 40.0], andĀ ā1.2% decrease [ā6.9 to 5.9]; and neointimal volumeĀ ā14.4% decrease [ā29.2 toĀ ā9.5], andĀ ā15.8% decrease [ā38.1 to 28.3]. The FFR gradient along the treated stent (difference in FFR between the distal and the proximal stent edge) was 0.37Ā Ā±Ā 0.18 pre-procedure, 0.06 Ā± 0.04 post-procedure, and 0.05 Ā± 0.05 at follow-up. In all post-procedural OCT images, intrastent dissections were seen, which were sealed at follow-up OCT.ConclusionsDEB restore coronary blood flow by means of a short-term mechanical effect, causing anĀ increase in lumen and stent volumes and compression of neointimal hyperplasia (with intra-stent dissections). Due to the local drug effect, patency persists and may even improve at follow-up, withĀ further increase in lumen volume, decrease in neointimal volume, and complete sealing of neointimal dissections
Disturbed Presynaptic Ca2+ Signaling in Photoreceptors in the EAE Mouse Model of Multiple Sclerosis
Multiple sclerosis (MS) is a demyelinating disease caused by an auto-reactive immune system. Recent studies also demonstrated synapse dysfunctions in MS patients and MS mouse models. We previously observed decreased synaptic vesicle exocytosis in photoreceptor synapses in the EAE mouse model of MS at an early, preclinical stage. In the present study, we analyzed whether synaptic defects are associated with altered presynaptic Ca2+ signaling. Using high-resolution immunolabeling, we found a reduced signal intensity of Cav-channels and RIM2 at active zones in early, preclinical EAE. In line with these morphological alterations, depolarization-evoked increases of presynaptic Ca2+ were significantly smaller. In contrast, basal presynaptic Ca2+ was elevated. We observed a decreased expression of Na+/K+-ATPase and plasma membrane Ca2+ ATPase 2 (PMCA2), but not PMCA1, in photoreceptor terminals of EAE mice that could contribute to elevated basal Ca2+. Thus, complex Ca2+ signaling alterations contribute to synaptic dysfunctions in photoreceptors in early EAE
MicroRNA-targeting in spermatogenesis: Over-expressions of microRNA-23a/b-3p and its affected targeting of the genes ODF2 and UBQLN3 in spermatozoa of patients with oligoasthenozoospermia
Background
Male infertility is a multifactorial syndrome with diverse phenotypic representations. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in the post-transcriptional regulation of gene expression. Altered abundance levels of ODF2 and UBQLN3 have been reported in patients with different spermatogenic impairments. However, the transcriptional regulation of these two genes by miR-23a/b-3p is still unclear.
Objectives
To investigate experimentally whether miR-23a/b-3p targets the genes ODF2 and UBQLN3 and whether this targeting impacts abundance levels of ODF2 and UBQLN3 in patients with oligoasthenozoospermia.
Materials and methods
A total of 92 men attending a fertility clinic were included in the study, including 46 oligoasthenozoospermic men and 46 age-matched normozoospermic volunteers who served as controls. Reverse transcription-quantitative PCR (RT-qPCR), Western blot, and dual-luciferase (Firefly-Renilla) assays were used to validate the miRNAs and their target genes.
Results
RT-qPCR revealed that miR-23a/b-3p was more abundant and ODF2 and UBQLN3 targets were less abundant in men with impaired spermatogenesis. Besides, Western blot shows that ODF2 and UBQLN3 protein levels were reduced in men with impaired spermatogenesis. In silico prediction and dual-luciferase assays revealed that potential links exist between the higher abundance level of miR-23a/b-3p and the lower abundance level of ODF2 and UBQLN3 targets. Mutations in the miR-23a/b-3p-binding site within the 3ĖUTRs (3Ėuntranslated regions) of ODF2 and UBQLN3 genes resulted in abrogated responsiveness to miR-23a/b-3p. Correlation analysis showed that sperm count, motility, and morphology were negatively correlated with miR-23a/b-3p and positively correlated with the lower abundance level of UBQLN3, while ODF lower abundance level was positively correlated with sperm motility.
Conclusion
Findings indicate that the higher abundance level of miR-23a/b-3p and the lower abundance level of ODF2 and UBQLN3 targets are associated with oligoasthenozoospermia and male subfertility
Control of Insulin Release by Transient Receptor Potential Melastatin 3 (TRPM3) Ion Channels
Background/Aims: The release of insulin in response to increased levels of glucose in the blood strongly depends on Ca2+ influx into pancreatic beta cells by the opening of voltage-gated Ca2+ channels. Transient Receptor Potential Melastatin 3 proteins build Ca2+ permeable, non-selective cation channels serving as pain sensors of noxious heat in the peripheral nervous system. TRPM3 channels are also strongly expressed in pancreatic beta cells that respond to the TRPM3 agonist pregnenolone sulfate with Ca2+ influx and increased insulin release. Therefore, we hypothesized that in beta cells TRPM3 channels may contribute to pregnenolone sulfate- as well as to glucose-induced insulin release. Methods: We used INS-1 cells as a beta cell model in which we analysed the occurrence of TRPM3 isoformes by immunoprecipitation and western blotting and by cloning of RT-PCR amplified cDNA fragments. We applied pharmacological as well as CRISPR/Cas9-based strategies to analyse the interplay of TRPM3 and voltage-gated Ca2+ channels in imaging experiments (FMP, Fura-2) and electrophysiological recordings. In immunoassays, we examined the contribution of TRPM3 channels to pregnenolone sulfate- and glucose-induced insulin release. To confirm our findings, we generated beta cell-specific Trpm3-deficient mice and compared their glucose clearance with the wild type in glucose tolerance tests. Results: TRPM3 channels triggered the activity of voltage-gated Ca2+ channels and both channels together contributed to insulin release after TRPM3 activation. Trpm3-deficient INS-1 cells lacked pregnenolone sulfate-induced Ca2+ signals just like the pregnenolone sulfate-induced insulin release. Both, glucose-induced Ca2+ signals and the glucose-induced insulin release were strongly reduced. Accordingly, Trpm3-deficient mice displayed an impaired decrease of the blood sugar concentration after intraperitoneal or oral administration of glucose. Conclusion: The present study suggests an important role for TRPM3 channels in the control of glucose-dependent insulin release
Optical Coherence Tomography Imaging of Acute Stent Thrombosis After Stentys Stent Implantation
IP3-dependent Ca2+ signals are tightly controlled by CavĪ²3, but not by CavĪ²1, 2 and 4
Independent of its function as a subunit of voltage-gated Ca2+ channels, the CavĪ²3 subunit desensitizes fibroblasts and pancreatic Ī²-cells to low concentrations of inositol-1,4,5-trisphosphate (IP3). This alters agonist-induced Ca2+ signaling and cellular functions, for example, insulin secretion and wound healing. A total of four CavĪ² subunits exist, CavĪ²1, CavĪ²2, CavĪ²3, and CavĪ²4. To investigate whether the other CavĪ² subunits, like CavĪ²3, can desensitize cells to IP3 and thereby modulate Ca2+ signaling, we expressed the cDNAs of CavĪ²1, CavĪ²2, CavĪ²3, and CavĪ²4 in COS-7 cells lacking endogenous CavĪ² proteins. ATP stimulation of these cells results in the release of Ca2+ from intracellular stores. This receptor-mediated Ca2+ release is significantly decreased by CavĪ²3 but not by CavĪ²1, CavĪ²2, and CavĪ²4. Electrophysiological recordings of voltage-dependent Ca2+ currents from fibroblasts show a small Ca2+ current, the amplitude of which is slightly but not significantly smaller in fibroblasts from CavĪ²2 gene-deficient animals than in fibroblasts from wild-type animals. Compared with fibroblasts from wild-type animals, Ca2+ release is not significantly increased in CavĪ²2-deficient fibroblasts, in contrast to Ca2+ release in CavĪ²3-deficient fibroblasts. In summary, our results show that desensitization of cells to low concentrations of IP3 is a specific property of CavĪ²3 that is not shared by other CavĪ² subunits
Identification of Inhibitory Ca2+ Binding Sites in the Upper Vestibule of the Yeast Vacuolar TRP Channel.
By vacuolar patch-clamp and Ca imaging experiments, we show that the yeast vacuolar transient receptor potential (TRPY) channel 1 is activated by cytosolic Ca and inhibited by Ca from the vacuolar lumen. The channel is cooperatively affected by vacuolar Ca (Hill coefficient, 1.5), suggesting that it may accommodate a Ca receptor that can bind two calcium ions. Alanine scanning of six negatively charged amino acid residues in the transmembrane S5 and S6 linker, facing the vacuolar lumen, revealed that two aspartate residues, 401 and 405, are essential for current inhibition and direct binding of Ca. Expressed in HEK-293 cells, a significant fraction of TRPY1, present in the plasma membrane, retained its Ca sensitivity. Based on these data and on homology with TRPV channels, we conclude that D401 and D405 are key residues within the vacuolar vestibule of the TRPY1 pore that decrease cation access or permeation after Ca binding