Patch clamp recording of neurons is a labor-intensive and time-consuming procedure. Here, we demonstrate a tool that fully automatically performs electrophysiological recordings in label-free tissue ...slices. The automation covers the detection of cells in label-free images, calibration of the micropipette movement, approach to the cell with the pipette, formation of the whole-cell configuration, and recording. The cell detection is based on deep learning. The model is trained on a new image database of neurons in unlabeled brain tissue slices. The pipette tip detection and approaching phase use image analysis techniques for precise movements. High-quality measurements are performed on hundreds of human and rodent neurons. We also demonstrate that further molecular and anatomical analysis can be performed on the recorded cells. The software has a diary module that automatically logs patch clamp events. Our tool can multiply the number of daily measurements to help brain research.
After introduction, the invasive bivalve dreissenids became key species in the biota of Lake Balaton, the largest shallow lake in Central Europe. The contribution of dreissenid soft tissue and shell, ...as biotic phases, in element distribution and its interaction with the water and upper sediment phases were examined in two basins with different trophic conditions in spring and autumn. Six metals (Ba, Cu, Fe, Mn, Pb, Zn) were detected in all investigated phases. In general, metals were abundant in the water and soft tissue in the eastern basin in spring, and in the sediment and shells in the western basin in autumn. This might be associated with the more urbanized surroundings in the eastern, and the enhanced organic matter production in the western basin. High relative shares of Ba, Cu, Mn, and Pb were associated with the water and shell samples, whereas high shares of Fe and Zn were noted in the soft mussel tissue and sediments. Results suggest that dynamics of metal uptake by dreissenids depend on the seasonal change in metabolic activity. Shell metal content is less changeable; shells might absorb metals from both the soft tissue and water phases. Metallothionein peptides, the scavengers of intracellular metals, were determined to be biomarkers of the bulk contaminants rather than only metals. The present study shows that invasive bivalves, with high abundance, filtering activity, and storing capacity can significantly contribute to element distribution in the shoreline of a shallow lake ecosystem.
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DOBA, EMUNI, FZAB, GEOZS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Concentrations of insulin in the brain are severalfold higher than blood plasma levels. Insulin in the brain regulates the metabolism, molecular composition, and cognitive performance of ...microcircuits and reduces food intake; cerebral insulin levels are altered in diabetes, aging, obesity, and Alzheimer's disease. Released by pancreatic β cells, insulin passes the blood-brain barrier, but sources of locally released insulin still remain unclear. We find that insulin is strongly expressed in GABAergic neurogliaform cells in the cerebral cortex of the rat detected by single-cell digital PCR. Focal application of glucose or glibenclamide to neurogliaform cells mimics the excitation suppressing effect of external insulin on local microcircuits via insulin receptors. Thus, neurogliaform cells might link GABAergic and insulinergic action in cortical microcircuits.
C-150 a Mannich-type curcumin derivative, exhibited pronounced cytotoxic effects against eight glioma cell lines at micromolar concentrations. Inhibition of cell proliferation by C-150 was mediated ...by affecting multiple targets as confirmed at transcription and protein level. C-150 effectively reduced the transcription activation of NFkB, inhibited PKC-alpha which are constitutively over-expressed in glioblastoma. The effects of C-150 on the Akt/ Notch signaling were also demonstrated in a Drosophila tumorigenesis model. C-150 reduced the number of tumors in Drosophila with similar efficacy to mitoxantrone. In an in vivo orthotopic glioma model, C-150 significantly increased the median survival of treated nude rats compared to control animals. The multi-target action of C-150, and its preliminary in vivo efficacy would render this curcumin analogue as a potent clinical candidate against glioblastoma.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract Diet-induced hypercholesterolemia leads to oxidative/nitrative stress and subsequent myocardial dysfunction. However, the regulatory role of microRNAs in this phenomenon is unknown. We aimed ...to investigate, whether hypercholesterolemia-induced myocardial microRNA alterations play a role in the development of oxidative/nitrative stress and in subsequent cardiac dysfunction. Male Wistar rats were fed with 2% cholesterol/0.25% cholate-enriched or standard diet for 12 weeks. Serum and tissue cholesterol levels were significantly elevated by cholesterol-enriched diet. Left ventricular end-diastolic pressure was significantly increased in cholesterol-fed rats both in vivo and in isolated perfused hearts, indicating diastolic dysfunction. Myocardial expression of microRNAs was affected by cholesterol-enriched diet as assessed by microarray analysis. MicroRNA-25 showed a significant down-regulation as detected by microarray analysis and QRT-PCR. In silico target prediction revealed NADPH oxidase 4 (NOX4) as a putative target of microRNA-25. NOX4 protein showed significant up-regulation in the hearts of cholesterol-fed rats, while NOX1 and NOX2 remained unaffected. Cholesterol-feeding significantly increased myocardial oxidative/nitrative stress as assessed by dihydroethidium staining, protein oxidation assay, and nitro-tyrosine ELISA, respectively. Direct binding of microRNA-25 mimic to the 3′ UTR region of NOX4 was demonstrated using a luciferase reporter assay. Transfection of a microRNA-25 mimic into primary cardiomyocytes decreased superoxide production, while a microRNA-25 inhibitor resulted in an up-regulation of NOX4 protein and an increase in oxidative stress that was attenuated by the NADPH oxidase inhibitor diphenyleneiodonium. Here we demonstrated for the first time that hypercholesterolemia affects myocardial microRNA expression, and by down-regulating microRNA-25 increases NOX4 expression and consequently oxidative/nitrative stress in the heart. We conclude that hypercholesterolemia-induced microRNA alterations play an important role in the regulation of oxidative/nitrative stress and in consequent myocardial dysfunction.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
We aimed to characterize early changes in microRNA expression in acute cardioprotection by ischemic pre- and postconditioning in rat hearts. Hearts isolated from male Wistar rats were subjected to 1) ...time-matched nonischemic perfusion, 2) ischemia-reperfusion (30 min of coronary occlusion and 120 min of reperfusion), 3) preconditioning (3 × 5 min of coronary occlusion) followed by ischemia-reperfusion, or 4) ischemia-reperfusion with postconditioning (6 × 10 s of global ischemia-reperfusion at the onset of reperfusion). Infarct size was significantly reduced by both interventions. Of 350 different microRNAs assessed by microarray analysis, 147-160 microRNAs showed detectable expression levels. Compared with microRNA alterations induced by ischemia-reperfusion versus time-matched nonischemic controls, five microRNAs were significantly affected by both pre- and postconditioning (microRNA-125b*, microRNA-139-3p, microRNA-320, microRNA-532-3p, and microRNA-188), four microRNAs were significantly affected by preconditioning (microRNA-487b, microRNA-139-5p, microRNA-192, and microRNA-212), and nine microRNAs were significantly affected by postconditioning (microRNA-1, microRNA let-7i, microRNA let-7e, microRNA let-7b, microRNA-181a, microRNA-208, microRNA-328, microRNA-335, and microRNA-503). Expression of randomly selected microRNAs was validated by quantitative real-time PCR. By a systematic comparison of the direction of microRNA expression changes in all groups, we identified microRNAs, specific mimics, or antagomiRs that may have pre- and postconditioning-like cardioprotective effects (protectomiRs). Transfection of selected protectomiRs (mimics of microRNA-139-5p, microRNA-125b*, microRNA let-7b, and inhibitor of microRNA-487b) into cardiac myocytes subjected to simulated ischemia-reperfusion showed a significant cytoprotective effect. This is the first demonstration that the ischemia-reperfusion-induced microRNA expression profile is significantly influenced by both pre- and postconditioning, which shows the involvement of microRNAs in cardioprotective signaling. Moreover, by analysis of microRNA expression patterns in cardioprotection by pre- and postconditioning, specific protectomiRs can be revealed as potential therapeutic tools for the treatment of ischemia-reperfusion injury.
Little is known about the molecular mechanism including microRNAs (miRNA) in hypercholesterolemia-induced cardiac dysfunction. We aimed to explore novel hypercholesterolemia-induced pathway ...alterations in the heart by an unbiased approach based on miRNA omics, target prediction and validation. With miRNA microarray we identified forty-seven upregulated and ten downregulated miRNAs in hypercholesterolemic rat hearts compared to the normocholesterolemic group. Eleven mRNAs with at least 4 interacting upregulated miRNAs were selected by a network theoretical approach, out of which 3 mRNAs (beta-2 adrenergic receptor Adrb2, calcineurin B type 1 Ppp3r1 and calcium/calmodulin-dependent serine protein kinase Cask) were validated with qRT-PCR and Western blot. In hypercholesterolemic hearts, the expression of Adrb2 mRNA was significantly decreased. ADRB2 and PPP3R1 protein were significantly downregulated in hypercholesterolemic hearts. The direct interaction of Adrb2 with upregulated miRNAs was demonstrated by luciferase reporter assay. Gene ontology analysis revealed that the majority of the predicted mRNA changes may contribute to the hypercholesterolemia-induced cardiac dysfunction. In summary, the present unbiased target prediction approach based on global cardiac miRNA expression profiling revealed for the first time in the literature that both the mRNA and protein product of Adrb2 and PPP3R1 protein are decreased in the hypercholesterolemic heart.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Reactive oxygen species (ROS), antioxidants and their reduction-oxidation (redox) states all contribute to the redox homeostasis, but glutathione is considered to be the master regulator of it. We ...aimed to understand the relationship between the redox potential and the diverse glutathione transferase (GST) enzyme family by comparing the stress responses of two tomato cultivars (Solanum lycopersicum ‘Moneymaker’ and ‘Ailsa Craig’). Four-week-old plants were treated by two concentrations of mannitol, NaCl and salicylic acid. The lower H2O2 and malondialdehyde contents indicated higher stress tolerance of ‘Moneymaker’. The redox status of roots was characterized by measuring the reduced and oxidized form of ascorbate and glutathione spectrophotometrically after 24 h. The redox potential of ‘Ailsa Craig’ was more oxidized compared to ‘Moneymaker’ even under control conditions and became more positive due to treatments. High-throughput quantitative real-time PCR revealed that besides overall higher expression levels, SlGSTs were activated more efficiently in ‘Moneymaker’ due to stresses, resulting in generally higher GST and glutathione peroxidase activities compared to ‘Ailsa Craig’. The expression level of SlGSTs correlated differently, however Pearson's correlation analysis showed usually strong positive correlation between SlGST transcription and glutathione redox potential. The possible redox regulation of SlGST expressions was discussed.
•‘Moneymaker’ roots have more negative glutathione redox potential than ‘Ailsa Craig’.•HT-qPCR analysis showed overall higher SlGST expression levels in ‘Moneymaker’.•Most of SlGSTs were induced, both stress- and genotypic specificities were found.•Correlations between SlGST expression and redox state are stronger in ‘Ailsa Craig’.•GSTs may have redox status-directed feedback in their transcriptional regulation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Here we examined myocardial microRNA (miRNA) expression profile in a sensory neuropathy model with cardiac diastolic dysfunction and aimed to identify key mRNA molecular targets of the differentially ...expressed miRNAs that may contribute to cardiac dysfunction.
Male Wistar rats were treated with vehicle or capsaicin for 3 days to induce systemic sensory neuropathy. Seven days later, diastolic dysfunction was detected by echocardiography, and miRNAs were isolated from the whole ventricles.
Out of 711 known miRNAs measured by miRNA microarray, the expression of 257 miRNAs was detected in the heart. As compared to vehicle-treated hearts,
,
,
,
,
,
, and
were downregulated, while
was upregulated as validated also by quantitative real time polymerase chain reaction (qRT-PCR). By an in silico network analysis, we identified common mRNA targets (insulin-like growth factor 1 (
), solute carrier family 2 facilitated glucose transporter member 12 (
), eukaryotic translation initiation factor 4e
), and Unc-51 like autophagy activating kinase 2 (
)) targeted by at least three altered miRNAs. Predicted upregulation of these mRNA targets were validated by qRT-PCR.
This is the first demonstration that sensory neuropathy affects cardiac miRNA expression network targeting
,
,
, and
, which may contribute to cardiac diastolic dysfunction. These results further support the need for unbiased omics approach followed by in silico prediction and validation of molecular targets to reveal novel pathomechanisms.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Salicylic acid (SA) plays a crucial role not only in defence against pathogen attacks, but also in abiotic stress responses. Recently, some key steps of SA signalling outlined the importance of redox ...state-dependent processes. This study explores the role of glutathione transferases (GSTs) in the transcriptional reprogramming of redox status-related genes in seven-day-old wild type and
Atgst
mutant
Arabidopsis thaliana
plants. The timing of redox changes, detected by the redox-sensitive green fluorescent protein (roGFP2), differed in wild type roots treated with 10 μM or 100 μM SA. Our results verified how the applied SA concentrations had different effect on the expression of oxidative stress- and redox-related genes, among them on the expression of
AtGSTF8
and
AtGSTU19
genes. Lower vitality and less negative
E
GSH
values were specific characteristics of the
Atgst
mutants compared to the wild type plants throughout the experiment. Changes in the redox potential were only modest in the mutants after SA treatments. A slightly modified gene expression pattern was observed in control conditions and after 1 h of SA treatments in
Atgst
mutants compared to Col-0 roots. These data originating from the whole roots provide indirect evidence for the role of the investigated AtGSTF8 and AtGSTU19 isoenzymes in the transduction of the redox signal. Our results demonstrate that the investigated Arabidopsis GSTs have a role in maintaining the levels of reactive oxygen species- and redox homeostasis and are involved in transcriptional reprogramming in the roots.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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