Indicator Of Membrane Potential Research Articles

Effect of titanium dioxide nanoparticle (TiO2-NP) exposure in a novel Amur sturgeon Acipenser schrenckii hepatocyte cell line.

In vitro cell culture is crucial for predicting the toxicity of titanium dioxide nanoparticle (TiO2-NP). However, assessing the toxicity of TiO2-NPs in sturgeon remains difficult given the lack of sufficient cell lines. We established and characterized the first hepatocyte cell line from Acipenser schrenckii liver tissue (ASL). This ASL cell line proliferated well in Dulbecco's modified Eagle's medium at 25°C and 10% fetal bovine serum. ASL cells with a chromosome number of 244 were successfully transfected with the pEGFP-N3 plasmid. The ASL cell line's origin was verified as A. schrenckii through mitochondrial cytochrome C oxidase I and mitochondrial 16S ribosomal RNA (rRNA) sequencing. Using the ASL cell line as an in vitro model, we found that TiO2-NP exposure decreased the viability and promoted the damage of ASL cells (96-h LC50 = 331.8 μg mL-1). Increased reactive oxygen species and malondialdehyde levels in ASL cells suggested oxidative stress under TiO2-NP exposure. We also observed dysregulation of aspartate aminotransferase and alanine aminotransferase levels. By detecting calcium ions and mitochondrial membrane potential indicators, we found that the apoptotic pathway induced by endoplasmic reticulum stress played a major role at low concentrations of TiO2-NP-induced stress. Both mitochondria-mediated and endoplasmic reticulum stress promoted apoptosis under increasing TiO2-NP concentrations. In conclusion, the ASL cell line established in this study is a useful in vitro model for toxicological studies of TiO2-NP exposure in fish.

A new model of ischemia/reperfusion injury using precision-cut liver slices

Background: Primary cells isolated from the liver are widely used to investigate hepatic pathophysiology. However, these cells do not represent cell-to-cell interactions, three dimensional architecture, and zonal structure in the liver. Furthermore, characterization of cellular and molecular events during ischemia/reperfusion (I/R) is challenging in liver biopsies. Thus, a new model of I/R injury resembling the native liver is needed. Methods: We generated precision-cut liver slices (PCLS) from mouse and human livers using a Krumdieck automatic tissue slicer (Alabama R & D, Munford, AL). Thickness of individual PLCL was 200 microns. To simulate anoxia, nutrient depletion, and tissue acidosis during ischemia, PCLS were incubated in Krebs-Ringer-N-(2-Hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES) solution at pH 6.2 in the anaerobic chamber (Coy Laboratory, Grass Lake, MI) for 30 min. To simulate normoxia, nutrient repletion, and restoration to normal pH during reperfusion, ischemic PCLS were aerobically incubated in Dulbecco's modified eagle medium (DMEM) at pH 7.4 for up to 4 h. Changes in tissue viability were assessed with the release of lactate dehydrogenase (LDH) into the extracellular medium. Some PCLS were labeled with green fluorescent rhodamine-123 (Rd-123, mitochondrial membrane potential indicator) and red fluorescent propidium iodide (PI, necrosis marker). Confocal images of PCLS were collected after I/R. Results: LDH release assay revealed that while PCLS from chow diet (CD)-fed lean mice tolerated I/R injury well, those from high fat diet (HFD)-fed steatotic mice were susceptible to I/R injury. Confocal microscopy showed substantial mitochondrial depolarization (loss of Rd-123) and necrosis (nuclear labeling of PI) in the HFD group, which was, however, minimally observed in the CD group. Of importance, mitochondrial dysfunction and cell death after reperfusion occurred predominantly in hepatocytes. Heightened I/R injury in steatotic livers was also confirmed in primary hepatocytes isolated from either CD- or HFD-fed mice. To compare the similarities and differences between mouse and human livers, PCLS were generated from discarded human livers deemed unsuitable for transplant and subjected to 30 min of in vitro ischemia. The loss of mitochondrial membrane potential and hepatocyte death progressively increased after I/R in discarded human livers. At 1 h after reperfusion, diffusive fluorescence of Rd-123 and nuclear labeling of PI became evident, indicative of mitochondrial dysfunction and tissue injury after I/R. SUMMARY: LDH assay and confocal analysis of PCLS demonstrated heightened susceptibility of steatotic livers to I/R injury. In contrast to PCLS from lean livers, those from steatotic livers rapidly developed mitochondrial depolarization and onset of necrosis, cardinal features of I/R injury, even after short ischemia. The early phase of reperfusion injury in steatotic livers occurred predominantly in parenchymal cells. CONCLUSION: In vitro I/R with PCLS is a model suitable for hepatic I/R injury. This work (J-S K) was funded by the National Institutes of Health (DK079878), Mid-America Transplant Foundation (07201903), Foundation for Barnes-Jewish Hospital (4776, 5153), and Washington University Department of Surgery Pilot Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pharmacological Inhibition of LRRK2 Exhibits Neuroprotective Activity in Mouse Photothrombotic Stroke Model.

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common cause of Parkinson's disease (PD). Interestingly, recent studies have reported an increased risk of stroke in patients with PD harboring LRRK2 mutations, but there is no evidence showing the functional involvement of LRRK2 in stroke. Here, we found that LRRK2 kinase activity was significantly induced in the Rose-Bengal (RB) photothrombosis-induced stroke mouse model. Interestingly, stroke infarct volumes were significantly reduced, and neurological deficits were diminished by pharmacological inhibition of LRRK2 kinase activity using MLi-2, a brain-penetrant LRRK2 kinase inhibitor. Immunohistochemical analysis showed p-LRRK2 level in stroke lesions, co-localizing with mitophagy-related proteins (PINK, Parkin, LC3B, cytochrome c), suggesting their involvement in stroke progression. Overlapping p-LRRK2 with cytochrome c/TUNEL/JC-1 (an indicator of mitochondrial membrane potential) puncta in RB photothrombosis indicated LRRK2-induced mitochondrial apoptosis, which was blocked by MLi-2. These results suggest that pharmacological inhibition of LRRK2 kinase activity could attenuate mitochondrial apoptosis, ultimately leading to neuroprotective potential in stroke progression. In conclusion, LRRK2 kinase activity might be neuro-pathogenic due to impaired mitophagy in stroke progression, and pharmacological inhibition of LRRK2 kinase activity could be beneficial in reducing the risk of stroke in patients with LRRK2 mutations.

Thioflavin T indicates mitochondrial membrane potential in mammalian cells

The fluorescent benzothiazole dye thioflavin T (ThT) is widely used as a marker for protein aggregates, most commonly in the context of neurodegenerative disease research and diagnosis. Recently, this same dye was shown to indicate membrane potential in bacteria due to its cationic nature. This finding prompted a question whether ThT fluorescence is linked to the membrane potential in mammalian cells, which would be important for appropriate utilization of ThT in research and diagnosis. Here, we show that ThT localizes into the mitochondria of HeLa cells in a membrane-potential-dependent manner. Specifically, ThT colocalized in cells with the mitochondrial membrane potential indicator tetramethylrhodamine methyl ester (TMRM) and gave similar temporal responses as TMRM to treatment with a protonophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). Additionally, we found that presence of ThT together with exposure to blue light (λ = 405 nm), but neither factor alone, caused depolarization of mitochondrial membrane potential. This additive effect of the concentration and blue light was recapitulated by a mathematical model implementing the potential-dependent distribution of ThT and its effect on mitochondrial membrane potential through photosensitization. These results show that ThT can act as a mitochondrial membrane potential indicator in mammalian cells, when used at low concentrations and with low blue light exposure. However, it causes dissipation of the mitochondrial membrane potential depending additively on its concentrations and blue light exposure. This conclusion motivates a re-evaluation of ThT’s use at micromolar range in live-cell analyses and indicates that this dye can enable future studies on the potential connections between mitochondrial membrane potential dynamics and protein aggregation.

Characterization of the far-red fluorescent probe MitoView 633 for dynamic mitochondrial membrane potential measurement.

Introduction: MitoView 633, a far-red fluorescent dye, exhibits the ability to accumulate within mitochondria in a membrane potential-dependent manner, as described by the Nernst equation. This characteristic renders it a promising candidate for bioenergetics studies, particularly as a robust indicator of mitochondrial membrane potential (DYm). Despite its great potential, its utility in live cell imaging has not been well characterized. Methods: This study seeks to characterize the spectral properties of MitoView 633 in live cells and evaluate its mitochondrial staining, resistance to photobleaching, and dynamics during DYm depolarization. The co-staining and imaging of MitoView 633 with other fluorophores such as MitoSOX Red and Fluo-4 AM were also examined in cardiomyocytes using confocal microscopy. Results and Discussion: Spectrum analysis showed that MitoView 633 emission could be detected at 660 ± 50nm, and exhibited superior thermal stability compared to tetramethylrhodamine methyl ester (TMRM), a commonly used DYm indicator, which emits at 605 ± 25nm. Confocal imaging unequivocally illustrated MitoView 633's specific localization within the mitochondrial matrix, corroborated by its colocalization with MitoTracker Green, a well-established mitochondrial marker. Furthermore, our investigation revealed that MitoView 633 exhibited minimal photobleaching at the recommended in vitro concentrations. Additionally, the dynamics of MitoView 633 fluoresce during carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, a mitochondrial uncoupler)-induced DYm depolarization mirrored that of TMRM. Importantly, MitoView 633 demonstrated compatibility with co-staining alongside MitoSOX Red and Fluo-4 AM, enabling concurrent monitoring of DYm, mitochondrial ROS, and cytosolic Ca2+ in intact cells. Conclusion: These findings collectively underscore MitoView 633 as a superb molecular probe for the singular or combined assessment of DYm and other indicators in live cell imaging applications.

Accurate quantification of total bacteria in raw milk by flow cytometry using membrane potential as a key viability parameter

Total bacterial count (TBC) is one of the most widely accepted indicators to evaluate raw milk quality. Rapidly quantifying TBC, by flow cytometry (FCM) coupled with viability indicators, is increasingly being preferred over time-consuming cultivation-based methods. However, whether viability indicators accurately identify viable bacteria, challenged by natural stresses, remains largely unexplored. Here, three typical milk-borne species were challenged with representative dairy farm disinfection conditions to identify the optimal viability parameter through multiparameter FCM, which has not been attempted so far. We demonstrated that only the membrane potential accurately distinguished between viable and dead cells under stress and exhibited considerable correlation with the plate counting method, despite that all viability parameters recognized highly active and dead cells. The membrane potential indicator, DiBAC4(3), was first combined with SYTO64 to establish a novel FCM-based method, while formulating a conversion equation for the FCM-plate counting results. Moreover, the performance of the developed method was verified using real samples, where TBC in raw milk was accurately quantified within 2 h. Together, our results demonstrated that membrane potential optimally reflected the bacterial viability in raw milk under stress and emphasized that the viability of stressed cells should be carefully identified to quantify viable bacteria through FCM.

A new fluorescent sensor mitoferrofluor indicates the presence of chelatable iron in polarized and depolarized mitochondria

Mitochondrial chelatable iron contributes to the severity of several injury processes, including ischemia/reperfusion, oxidative stress, and drug toxicity. However, methods to measure this species in living cells are lacking. To measure mitochondrial chelatable iron in living cells, here we synthesized a new fluorescent indicator, mitoferrofluor (MFF). We designed cationic MFF to accumulate electrophoretically in polarized mitochondria, where a reactive group then forms covalent adducts with mitochondrial proteins to retain MFF even after subsequent depolarization. We also show in cell-free medium that Fe2+ (and Cu2+), but not Fe3+, Ca2+, or other biologically relevant divalent cations, strongly quenched MFF fluorescence. Using confocal microscopy, we demonstrate in hepatocytes that red MFF fluorescence colocalized with the green fluorescence of the mitochondrial membrane potential (ΔΨm) indicator, rhodamine 123 (Rh123), indicating selective accumulation into the mitochondria. Unlike Rh123, mitochondria retained MFF after ΔΨm collapse. Furthermore, intracellular delivery of iron with membrane-permeant Fe3+/8-hydroxyquinoline (FeHQ) quenched MFF fluorescence by ∼80% in hepatocytes and other cell lines, which was substantially restored by the membrane-permeant transition metal chelator pyridoxal isonicotinoyl hydrazone. We also show FeHQ quenched the fluorescence of cytosolically coloaded calcein, another Fe2+ indicator, confirming that Fe3+ in FeHQ undergoes intracellular reduction to Fe2+. Finally, MFF fluorescence did not change after addition of the calcium mobilizer thapsigargin, which shows MFF is insensitive to physiologically relevant increases of mitochondrial Ca2+. In conclusion, the new sensor reagent MFF fluorescence is an indicator of mitochondrial chelatable Fe2+ in normal hepatocytes with polarized mitochondria as well as in cells undergoing loss of ΔΨm.

Erratum: Pinion et al., "Differential Electrographic Signatures Generated by Mechanistically-Diverse Seizurogenic Compounds in the Larval Zebrafish Brain".

<h3>Objective</h3> Patients with orthostatic hypotension may experience neck pain radiating to the occipital region of the skull and the shoulders while standing (so-called coat-hanger ache). This study assessed muscle membrane potential in the trapezius muscle of patients with orthostatic hypotension and healthy subjects during head-up tilt (HUT), by measuring velocity recovery cycles (VRCs) of muscle action potentials as an indicator of muscle membrane potential. <h3>Methods</h3> Eight patients with multiple system atrophy (MSA), orthostatic hypotension and a positive history for coat-hanger pain and eight normal controls (NCs) were included in this study. Repeated VRCs were recorded from the trapezius muscle by direct muscle stimulation in the supine position and during HUT for 10 min. <h3>Results</h3> Muscle VRC recordings did not differ between MSA patients and NCs in the supine position. During HUT, early supernormality decreased progressively and relative refractory period increased in MSA patients whereas VRC measures remained unchanged in NCs. Ten minutes after the start of HUT, early supernormality was reduced by 44% and relative refractory period was increased by 17%. <h3>Conclusions</h3> Muscle membranes in patients with orthostatic hypotension become progressively depolarised during standing. Membrane depolarisation is most likely the result of muscle ischaemia, related to the drop in perfusion pressure caused by orthostatic hypotension. Coat-hanger ache is most likely a consequence of this muscle ischaemia.

Inhibiting Succinate Release Worsens Cardiac Reperfusion Injury by Enhancing Mitochondrial Reactive Oxygen Species Generation.

BackgroundThe metabolite succinate accumulates during cardiac ischemia. Within 5 minutes of reperfusion, succinate returns to baseline levels via both its release from cells and oxidation by mitochondrial complex II. The latter drives reactive oxygen species (ROS) generation and subsequent opening of the mitochondrial permeability transition (PT) pore, leading to cell death. Targeting succinate dynamics (accumulation/oxidation/release) may be therapeutically beneficial in cardiac ischemia–reperfusion (IR) injury. It has been proposed that blocking MCT1 (monocarboxylate transporter 1) may be beneficial in IR injury, by preventing succinate release and subsequent engagement of downstream inflammatory signaling pathways. In contrast, herein we hypothesized that blocking MCT1 would retain succinate in cells, exacerbating ROS generation and IR injury.Methods and ResultsUsing the mitochondrial ROS probe mitoSOX and a custom‐built murine heart perfusion rig built into a spectrofluorometer, we measured ROS generation in situ during the first moments of reperfusion. We found that acute MCT1 inhibition enhanced mitochondrial ROS generation at reperfusion and worsened IR injury (recovery of function and infarct size). Both of these effects were abrogated by tandem inhibition of mitochondrial complex II, suggesting that succinate retention worsens IR because it drives more mitochondrial ROS generation. Furthermore, using the PT pore inhibitor cyclosporin A, along with monitoring of PT pore opening via the mitochondrial membrane potential indicator tetramethylrhodamine ethyl ester, we herein provide evidence that ROS generation during early reperfusion is upstream of the PT pore, not downstream as proposed by others. In addition, pore opening was exacerbated by MCT1 inhibition.ConclusionsTogether, these findings highlight the importance of succinate dynamics and mitochondrial ROS generation as key determinants of PT pore opening and IR injury outcomes.

The Short-Term Opening of Cyclosporin A-Independent Palmitate/Sr2+-Induced Pore Can Underlie Ion Efflux in the Oscillatory Mode of Functioning of Rat Liver Mitochondria.

Mitochondria are capable of synchronized oscillations in many variables, but the underlying mechanisms are still unclear. In this study, we demonstrated that rat liver mitochondria, when exposed to a pulse of Sr2+ ions in the presence of valinomycin (a potassium ionophore) and cyclosporin A (a specific inhibitor of the permeability transition pore complex) under hypotonia, showed prolonged oscillations in K+ and Sr2+ fluxes, membrane potential, pH, matrix volume, rates of oxygen consumption and H2O2 formation. The dynamic changes in the rate of H2O2 production were in a reciprocal relationship with the respiration rate and in a direct relationship with the mitochondrial membrane potential and other indicators studied. The pre-incubation of mitochondria with Ca2+(Sr2+)-dependent phospholipase A2 inhibitors considerably suppressed the accumulation of free fatty acids, including palmitic and stearic acids, and all spontaneous Sr2+-induced cyclic changes. These data suggest that the mechanism of ion efflux from mitochondria is related to the opening of short-living pores, which can be caused by the formation of complexes between Sr2+(Ca2+) and endogenous long-chain saturated fatty acids (mainly, palmitic acid) that accumulate due to the activation of phospholipase A2 by the ions. A possible role for transient palmitate/Ca2+(Sr2+)-induced pores in the maintenance of ion homeostasis and the prevention of calcium overload in mitochondria under pathophysiological conditions is discussed.

In vitro assessment of stearyl triphenyl phosphonium toxicity in drug-resistant tumor cells

Introduction: The triphenyl phosphonium residue is a well-documented mitochondriotropic that has been shown to improve the accumulation of biomolecules in mitochondria. Stearyl triphenyl phosphonium (STPP) modified liposomes have been shown to facilitate the selective accumulation of various biomolecules in mitochondria resulting in improved effect in-vitro and in-vivo. More recently, STPP was reported to have higher toxicity towards a drug resistant ovarian cancer cell line compare to a non-drug resistant cell line. The purpose of this study was to further investigate STPP toxicity using multiple drug resistant and non-drug resistant cell lines. Methods: STPP was incorporated into phosphatidylcholine cholesterol liposomes using the thin film hydration method. Mean particle size and zeta potential was measured using dynamic light scattering. The 5,5,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimi-dazoylcarbocyanine iodide (JC-1) dye accumulation assay was used as an indicator of mitochondrial membrane potential in the tested cell lines. Cytotoxicity of the preparations towards different cell lines was determined using light microscopy and the CellTiter 96® AQueous One Solution Cell Proliferation assay. Results: The JC-1 accumulation assay confirmed that the drug-resistant cell lines had significantly higher dye accumulation than the non-drug resistant cell lines. Higher cytotoxicity of STPP towards drug resistant cell line was seen when incorporated into liposomes but not when dissolved in dimethyl sulfoxide (DMSO). STPP showed a comparable toxicity profile to the known oxidative phosphorylation uncoupler carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone (FCCP). Discussion: Taken together, the data suggest that higher STPP toxicity in the drug-resistant cell lines is influenced by the presence of liposomal lipids and that STPP acts in a way similar to an oxidative phosphorylation uncoupler and is therefore more toxic to the drug-resistant cells that rely on a higher mitochondrial membrane potential to maintain their viability.

Mitochondrial Membrane Potential Assay.

Mitochondrial function, a key indicator of cell health, can be assessed through monitoring changes in mitochondrial membrane potential (MMP). Cationic fluorescent dyes are commonly used tools to assess MMP. We used a water-soluble mitochondrial membrane potential indicator (m-MPI) to detect changes in MMP in various types of cells, such as HepG2, HepaRG, and AC16 cells. A homogenous cell-based MMP assay has been optimized and performed in a 1536-well plate format, which can be used to screen several compound libraries for mitochondrial toxicity by evaluating the effects of chemical compounds on MMP.

A NIR Emitting Cyanine with Large Stokes' Shift for Mitochondria and Identification of their Membrane Potential Disruption.

An NIR emitting (λem ≈730 nm) cyanine probe ExCy was synthesized in good yields by extending the π-conjugation length (i. e., with furan moiety) to the donor-accepter system. ExCy exhibited a large Stokes' shift (Δλ≈100 nm) due to strong intramolecular charge transfer (ICT), and high fluorescence quantum yield (Φfl ≈0.47 in DCM). Due to its low fluorescence in an aqueous environment (Φfl ≈0.007 in H2 O), the probe exhibited the potential of achieving a large fluorescence turn-on upon entering a hydrophobic cellular environment. Fluorescence confocal microscopy studies revealed that ExCy was readily excitable with a far-red laser line (i. e., 640 nm) while the corresponding emission was collected in the NIR region. ExCy exhibited excellent selectivity towards live cell mitochondria according to the co-localization studies. The probe also exhibited high photostability, long-term imaging ability and wash-free staining ability, when being applied to live cells. Our studies indicated that the mitochondrial localization of ExCy was dependent on the membrane potential of the mitochondria. ExCy was successfully utilized as a mitochondrial membrane potential dysfunction indicator to visually identify cells with mitochondrial dysfunction via fluorescence confocal microscopy. ExCy was further examined for potential in vivo imaging of zebrafish.

An Autonomous Molecular Bioluminescent Reporter (AMBER) for Voltage Imaging in Freely Moving Animals.

Genetically encoded reporters have greatly increased our understanding of biology. While fluorescent reporters have been widely used, photostability and phototoxicity have hindered their use in long-term experiments. Bioluminescence overcomes some of these challenges but requires the addition of an exogenous luciferin limiting its use. Using a modular approach, Autonomous Molecular BioluminEscent Reporter (AMBER), an indicator of membrane potential is engineered. Unlike other bioluminescent systems, AMBER is a voltage-gated luciferase coupling the functionalities of the Ciona voltage-sensing domain (VSD) and bacterial luciferase, luxAB. When co-expressed with the luciferin-producing genes, AMBER reversibly switches the bioluminescent intensity as a function of membrane potential. Using biophysical and biochemical methods, it is shown that AMBER switches its enzymatic activity from an OFF to an ON state as a function of the membrane potential. Upon depolarization, AMBER switches from a low to a high enzymatic activity state, showing a several-fold increase in the bioluminescence output (ΔL/L). AMBER in the pharyngeal muscles and mechanosensory touch neurons of Caenorhabditis elegans is expressed. Using the compressed sensing approach, the electropharingeogram of the C. elegans pharynx is reconstructed, validating the sensor in vivo. Thus, AMBER represents the first fully genetically encoded bioluminescent reporter without requiring exogenous luciferin addition.

Live Imaging of the Mitochondrial Glutathione Redox State in Primary Neurons using a Ratiometric Indicator.

Mitochondrial redox homeostasis is important for neuronal viability and function. Although mitochondria contain several redox systems, the highly abundant thiol-disulfide redox buffer glutathione is considered a central player in antioxidant defenses. Therefore, measuring the mitochondrial glutathione redox potential provides useful information about mitochondrial redox status and oxidative stress. Glutaredoxin1-roGFP2 (Grx1-roGFP2) is a genetically encoded, green fluorescent protein (GFP)-based ratiometric indicator of the glutathione redox potential that has two redox-state-sensitive excitation peaks at 400 nm and 490 nm with a single emission peak at 510 nm. This article describes how to perform confocal live microscopy of mitochondria-targeted Grx1-roGFP2 in primary hippocampal and cortical neurons. It describes how to assess steady-state mitochondrial glutathione redox potential (e.g., to compare disease states or long-term treatments) and how to measure redox changes upon acute treatments (using the excitotoxic drug N-methyl-D-aspartate (NMDA) as an example). In addition, the article presents co-imaging of Grx1-roGFP2 and the mitochondrial membrane potential indicator, tetramethylrhodamine, ethyl ester (TMRE), to demonstrate how Grx1-roGPF2 can be multiplexed with additional indicators for multiparametric analyses. This protocol provides a detailed description of how to (i) optimize confocal laser scanning microscope settings, (ii) apply drugs for stimulation followed by sensor calibration with diamide and dithiothreitol, and (iii) analyze data with ImageJ/FIJI.

A dual optical probe with larger stokes shift for simultaneous detection of Cu2+ and Zn2+ ions and aggregation induced enhanced emission empowering selective detection of Cu2+ ions

A new aggregation induced enhanced emission (AIEE) active probe (1) has been synthesized via single step condensation reaction of 2-amino-3-benzyloxypyridine with 3-allylsalicylaldehyde in methanol. Probe 1 has been fully characterized by various spectroscopic techniques i.e. IR, NMR (1H and 13C), HRMS and X-ray crystallography. 1 displays a large stokes shift of 190 nm. It recognizes Cu2+ ions colorimetrically and fluorescence intensity enhances on the addition of Zn2+ ions. 1 form nanoaggregates in H2O:CH3OH (80:20, v/v) which detects Cu2+ ions selectively. 1 in the presence of Zn2+ ions and its nanoaggregates n1 show switch on behaviour of green fluorescence comparable to mitochondrial membrane potential indicator, Mitotracker-Red, in larval gut tissues of Drosophila melanogaster.

ALTERATION OF MITOCHONDRIAL MEMBRANE POTENTIAL (ΔΨM): POTENTIAL ROLE IN THE MECHANISM OF MEHP‐INDUCED INHIBITION OF STEROID BIOSYNTHESIS IN MA‐10 LEYDIG CELLS

INTRODUCTION AND OBJECTIVESMono‐(2‐ethylhexyl) phthalate (MEHP) is the active metabolite of di‐(2‐ethylhexyl) phthalate (DEHP), a plasticizer with endocrine disruptor activity that is widely used in the manufacturing industry. MEHP inhibits luteinizing hormone (LH)‐stimulated steroid biosynthesis in Leydig cells by molecular mechanisms that remain unclear. In the present study, using MA‐10 mouse tumor Leydig cells, we examined changes in mitochondrial function caused by MEHP exposures in relationship to possible link to alteration of progesterone biosynthesis.METHODSThe effect of MEHP on LH‐stimulated progesterone production was assessed after treating MA‐10 cells with (0–300 μM) MEHP for 24 hrs. Progesterone production was quantified by enzyme‐linked immunosorbent assay (ELISA). To measure oxygen consumption, cells were treated with MEHP (0–300 μM) for 24 hrs, and oxygen consumption was monitored using a Clarke‐type oxygen electrode. Mitochondrial membrane potential (ΔΨm) was assessed using the JC‐1 dye mitochondrial membrane potential indicator. Mitochondria‐derived superoxide was measured using the MitoSOX™ red mitochondrial superoxide indicator.RESULTSExposures of MA‐10 cells to 0–300 μM MEHP caused inhibition LH‐stimulated progesterone biosynthesis in a dose‐dependent manner. The decrease in the Leydig ability to produce progesterone correlated with a loss in the Leydig cell mitochondrial membrane potential (ΔΨm), and was linked to perturbation of the capacity to energize the mitochondrial membrane. MEHP exposure was suggested to cause a disruption in the electron flow through the electron transport chain, evidenced by a decrease in the ability to consumption molecular oxygen and an increase in the generation of superoxide in the mitochondria.CONCLUSIONSTogether, these observations suggest that MEHP inhibits steroid biosynthesis in MA‐10 cells via a mechanism involving a loss of the mitochondrial membrane potential (ΔΨm) and an increase in the oxidative stress level, two crucial changes known to affect mitochondrial function and cause inhibition of STAR‐mediated cholesterol transport and steroid biosynthesis.Support or Funding InformationThis material is based upon work supported by NIH grant SC2GM099578 and by the North Carolina Biotechnology Center, NCBC grant #2019‐IDG‐1017.

Establishment of cardiac action potential recording using a membrane potential indicator in the mouse sinoatrial node

Establishment of cardiac action potential recording using a membrane potential indicator in the mouse sinoatrial node

Extracellular Mitochondrial Dysfunction in Cerebrospinal Fluid of Patients with Delayed Cerebral Ischemia after Aneurysmal Subarachnoid Hemorrhage.

Mitochondrial dysfunction is related to brain ischemic injury and neural cell death. However, little is known about the association between mitochondrial dysfunction of cerebrospinal fluid (CSF) and delayed cerebral ischemia (DCI) following subarachnoid hemorrhage (SAH). The objective of this study was to investigate whether extracellular CSF mitochondria might serve as a potential biomarker for DCI. CSF samples were serially collected at 1, 3, and 5days following SAH in 33 patients (DCI, n = 12; and non-DCI, n = 21) who underwent coil embolization. To monitor mitochondrial membrane potentials, JC-1 dye was used. The ratio (red/green) of JC-1 was considered as an indicator of intact mitochondrial membrane potential. Flow cytometry was done to analyze extracellular mitochondria particles and their possible cellular origins. DCI patients had lower JC-1 red/green ratios than non-DCI patients at 1day (3.35 [3.20-3.75] vs. 3.70 [3.40-3.95] in non-DCI) and 3days (4.65 [4.45-5.00] vs. 5.10 [4.65-5.30] in non-DCI) after SAH. At 5days after SAH, JC-1 red/green ratio was significantly lower in DCI than that in non-DCI (3.05 [2.90-3.35] vs. 4.20 [4.10-4.50]; p < 0.01) patients. DCI patients had a higher percentage of vWF-positive mitochondria (40.10% [38.25%-44.90%] vs. 30.20% [25.70%-36.68%]) and a lower percentage of GLAST-positive mitochondria particles (26.85% [17.10%-30.00%] vs. 31.60% [26.70%-35.00%]) than non-DCI patients. However, there was no significant difference in CD45-positive (p = 0.369) or CD41/61-positive mitochondrial particles (p = 0.155) between the two groups of patients. Mitochondrial membrane potential could be a marker of DCI. JC-1 ratios seemed to be able to predict future DCI onset. Further studies are needed to determine detailed mechanisms of extracellular mitochondria-mediated cell-to-cell signals in DCI.

Theranostic nanobubble encapsulating a plasmon-enhanced upconversion hybrid nanosystem for cancer therapy.

Nanobubble (NB), which simultaneously enhances ultrasound (US) images and access therapeutic platforms, is required for future cancer treatment.Methods: We designed a theranostic agent for novel cancer treatment by using an NB-encapsulated hybrid nanosystem that can be monitored by US and fluorescent imaging and activated by near-infrared (NIR) light. The nanosystem was transported to the tumor through the enhanced permeability and retention effect. The hybrid nanosystem comprised upconversion nanoparticle (UCNP) and mesoporous silica-coated gold nanorod (AuNR@mS) with the photosensitizer merocyanine 540 to realize dual phototherapy.Results: With the NIR light-triggered, the luminous intensity of the UCNP was enhanced by doping holmium ion and emitted visible green and red lights at 540 and 660 nm. The high optical density state between the UCNP and AuNR@mS can induce plasmonic enhancement to improve the photothermal and photodynamic effects, resulting in cell death by apoptosis. The nanosystem showed excellent stability to avoid the aggregation of nanoparticles during the treatment. JC-1 dye was used as an indicator of mitochondrial membrane potential to identify the mechanism of cell death. The results of in vitro and in vivo analyses confirmed the curative effect of improved dual phototherapy.Conclusion: We developed and showed the therapeutic functions of a novel nanosystem with the combination of multiple theranostic nanoplatforms that can be triggered and activated by 808 nm NIR laser and US.