Light sources and cameras for standard in vitro
 membrane potential and high-speed ion imaging

Herein, we evaluated in vitro the anti-leishmanial activity of betulin derivatives in Venezuelan isolates of Leishmania amazonensis, isolated from patients with therapeutic failure. Methods: We analyzed promastigote in vitro susceptibility as well as the cytotoxicity and selectivity of the evaluated compounds. Additionally, the activity of selected compounds was determined in intracellular amastigotes. Finally, to gain hints on their potential mechanism of action, the effect of the most promising compounds on plasma and mitochondrial membrane potential, and nitric oxide and superoxide production by infected macrophages was determined. Results: From the tested 28 compounds, those numbered 18 and 22 were chosen for additional studies. Both 18 and 22 were active (GI50 ≤ 2 µM, cytotoxic CC50 > 45 µM, SI > 20) for the reference strain LTB0016 and for patient isolates. The results suggest that 18 significantly depolarized the plasma membrane potential (p < 0.05) and the mitochondrial membrane potential (p < 0.05) when compared to untreated cells. Although neither 18 nor 22 induced nitric oxide production in infected macrophages, 18 induced superoxide production in infected macrophages. Conclusion: Our results suggest that due to their efficacy and selectivity against intracellular parasites and the potential mechanisms underlying their leishmanicidal effect, the compounds 18 and 22 could be used as tools for designing new chemotherapies against leishmaniasis.

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Review of T-2307, an Investigational Agent That Causes Collapse of Fungal Mitochondrial Membrane Potential

Journal of Fungi ◽

10.3390/jof7020130 ◽

2021 ◽

Vol 7 (2) ◽

pp. 130

Author(s):

Nathan P. Wiederhold

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Candida Species ◽

Mammalian Cells ◽

Mitochondrial Membrane ◽

Fungal Infections ◽

Published Data ◽

Candida Auris

Invasive infections caused by Candida that are resistant to clinically available antifungals are of increasing concern. Increasing rates of fluconazole resistance in non-albicans Candida species have been documented in multiple countries on several continents. This situation has been further exacerbated over the last several years by Candida auris, as isolates of this emerging pathogen that are often resistant to multiple antifungals. T-2307 is an aromatic diamidine currently in development for the treatment of invasive fungal infections. This agent has been shown to selectively cause the collapse of the mitochondrial membrane potential in yeasts when compared to mammalian cells. In vitro activity has been demonstrated against Candida species, including C. albicans, C. glabrata, and C. auris strains, which are resistant to azole and echinocandin antifungals. Activity has also been reported against Cryptococcus species, and this has translated into in vivo efficacy in experimental models of invasive candidiasis and cryptococcosis. However, little is known regarding the clinical efficacy and safety of this agent, as published data from studies involving humans are not currently available.

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Application of Light-Emitting Diodes for Improving the Nutritional Quality and Bioactive Compound Levels of Some Crops and Medicinal Plants

Molecules ◽

10.3390/molecules26051477 ◽

2021 ◽

Vol 26 (5) ◽

pp. 1477

Author(s):

Woo-Suk Jung ◽

Ill-Min Chung ◽

Myeong Ha Hwang ◽

Seung-Hyun Kim ◽

Chang Yeon Yu ◽

...

Keyword(s):

Plant Growth ◽

In Vitro Culture ◽

Light Emitting Diodes ◽

Photosynthetic Pigment ◽

Antioxidant Properties ◽

Light Sources ◽

Light Emitting ◽

Cell Defense ◽

Led Irradiation

Light is a key factor that affects phytochemical synthesis and accumulation in plants. Due to limitations of the environment or cultivated land, there is an urgent need to develop indoor cultivation systems to obtain higher yields with increased phytochemical concentrations using convenient light sources. Light-emitting diodes (LEDs) have several advantages, including consumption of lesser power, longer half-life, higher efficacy, and wider variation in the spectral wavelength than traditional light sources; therefore, these devices are preferred for in vitro culture and indoor plant growth. Moreover, LED irradiation of seedlings enhances plant biomass, nutrient and secondary metabolite levels, and antioxidant properties. Specifically, red and blue LED irradiation exerts strong effects on photosynthesis, stomatal functioning, phototropism, photomorphogenesis, and photosynthetic pigment levels. Additionally, ex vitro plantlet development and acclimatization can be enhanced by regulating the spectral properties of LEDs. Applying an appropriate LED spectral wavelength significantly increases antioxidant enzyme activity in plants, thereby enhancing the cell defense system and providing protection from oxidative damage. Since different plant species respond differently to lighting in the cultivation environment, it is necessary to evaluate specific wavebands before large-scale LED application for controlled in vitro plant growth. This review focuses on the most recent advances and applications of LEDs for in vitro culture organogenesis. The mechanisms underlying the production of different phytochemicals, including phenolics, flavonoids, carotenoids, anthocyanins, and antioxidant enzymes, have also been discussed.

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Computational in Vitro Toxicology Uncovers Chemical Structures Impairing Mitochondrial Membrane Potential

Journal of Chemical Information and Modeling ◽

10.1021/acs.jcim.8b00433 ◽

2019 ◽

Vol 59 (2) ◽

pp. 702-712 ◽

Cited By ~ 4

Author(s):

David A. Dreier ◽

Nancy D. Denslow ◽

Christopher J. Martyniuk

Keyword(s):

Membrane Potential ◽

Mitochondrial Membrane Potential ◽

Mitochondrial Membrane ◽

In Vitro Toxicology ◽

Chemical Structures

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Turnover of the Active Fraction of IRS1 Involves Raptor-mTOR- and S6K1-Dependent Serine Phosphorylation in Cell Culture Models of Tuberous Sclerosis

Molecular and Cellular Biology ◽

10.1128/mcb.01254-05 ◽

2006 ◽

Vol 26 (17) ◽

pp. 6425-6434 ◽

Cited By ~ 119

Author(s):

O. Jameel Shah ◽

Tony Hunter

Keyword(s):

Cell Culture ◽

Tuberous Sclerosis ◽

High Speed ◽

Serine Phosphorylation ◽

Feedback Signal ◽

Insulin Receptor Substrates ◽

Igf I ◽

Culture Models ◽

Cell Culture Models

ABSTRACT The TSC1-TSC2/Rheb/Raptor-mTOR/S6K1 cell growth cassette has recently been shown to regulate cell autonomous insulin and insulin-like growth factor I (IGF-I) sensitivity by transducing a negative feedback signal that targets insulin receptor substrates 1 and 2 (IRS1 and -2). Using two cell culture models of the familial hamartoma syndrome, tuberous sclerosis, we show here that Raptor-mTOR and S6K1 are required for phosphorylation of IRS1 at a subset of serine residues frequently associated with insulin resistance, including S307, S312, S527, S616, and S636 (of human IRS1). Using loss- and gain-of-function S6K1 constructs, we demonstrate a requirement for the catalytic activity of S6K1 in both direct and indirect regulation of IRS1 serine phosphorylation. S6K1 phosphorylates IRS1 in vitro on multiple residues showing strong preference for RXRXXS/T over S/T,P sites. IRS1 is preferentially depleted from the high-speed pellet fraction in TSC1/2-deficient mouse embryo fibroblasts or in HEK293/293T cells overexpressing Rheb. These studies suggest that, through serine phosphorylation, Raptor-mTOR and S6K1 cell autonomously promote the depletion of IRS1 from specific intracellular pools in pathological states of insulin and IGF-I resistance and thus potentially in lesions associated with tuberous sclerosis.

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Identification of Positive Allosteric Modulators of Glycine Receptors from a High-Throughput Screen Using a Fluorescent Membrane Potential Assay

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057116657779 ◽

2016 ◽

Vol 21 (10) ◽

pp. 1042-1053 ◽

Cited By ~ 11

Author(s):

Clara Stead ◽

Adam Brown ◽

Cathryn Adams ◽

Sarah J. Nickolls ◽

Gareth Young ◽

...

Keyword(s):

Membrane Potential ◽

High Throughput ◽

Glycine Receptor ◽

Functional Characterization ◽

In Vitro Assays ◽

Allosteric Modulators ◽

Inhibitory Neurotransmission ◽

Starting Point ◽

Positive Allosteric Modulators

Glycine receptor 3 (GlyRα3) is a ligand-gated ion channel of the cys-loop family that plays a key role in mediating inhibitory neurotransmission and regulation of pain signaling in the dorsal horn. Potentiation of GlyRα3 function is therefore of interest as a putative analgesic mechanism with which to target new therapeutics. However, to date, positive allosteric modulators (PAMs) of this receptor with sufficient selectivity to enable target validation studies have not been described. To address this lack of pharmacological tools, we developed a suite of in vitro assays comprising a high-throughput fluorescent membrane potential screen and a medium-throughput electrophysiology assay using IonFlux HT together with conventional manual patch clamp. Using these assays, we conducted a primary screening campaign and report the structures of hit compounds identified as GlyR PAMs. Our functional characterization data reveal a hit compound with high efficacy relative to current known potentiators and selectivity over GABAAR, another major class of inhibitory neurotransmission receptors of importance to pain. These small-molecule GlyR PAMs have high potential both as early tool compounds to enable pharmacological studies of GlyR inhibitory neurotransmission and as a starting point for the development of potent, selective GlyRα3 PAMs as novel analgesics.

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Imaging the transmembrane and transendothelial sodium gradients in gliomas

Scientific Reports ◽

10.1038/s41598-021-85925-9 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Muhammad H. Khan ◽

John J. Walsh ◽

Jelena M. Mihailović ◽

Sandeep K. Mishra ◽

Daniel Coman ◽

...

Keyword(s):

Membrane Potential ◽

Magnetic Resonance ◽

Cell Membrane ◽

Normal Tissue ◽

Magnetic Resonance Spectroscopic Imaging ◽

Biological Factors ◽

High Sodium ◽

Intracellular Milieu

AbstractUnder normal conditions, high sodium (Na+) in extracellular (Na+e) and blood (Na+b) compartments and low Na+ in intracellular milieu (Na+i) produce strong transmembrane (ΔNa+mem) and weak transendothelial (ΔNa+end) gradients respectively, and these manifest the cell membrane potential (Vm) as well as blood–brain barrier (BBB) integrity. We developed a sodium (23Na) magnetic resonance spectroscopic imaging (MRSI) method using an intravenously-administered paramagnetic polyanionic agent to measure ΔNa+mem and ΔNa+end. In vitro 23Na-MRSI established that the 23Na signal is intensely shifted by the agent compared to other biological factors (e.g., pH and temperature). In vivo 23Na-MRSI showed Na+i remained unshifted and Na+b was more shifted than Na+e, and these together revealed weakened ΔNa+mem and enhanced ΔNa+end in rat gliomas (vs. normal tissue). Compared to normal tissue, RG2 and U87 tumors maintained weakened ΔNa+mem (i.e., depolarized Vm) implying an aggressive state for proliferation, whereas RG2 tumors displayed elevated ∆Na+end suggesting altered BBB integrity. We anticipate that 23Na-MRSI will allow biomedical explorations of perturbed Na+ homeostasis in vivo.

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Hyperpolarization-Activated Inward Current in Neurons of the Rat's Dorsal Nucleus of the Lateral Lemniscus In Vitro

Journal of Neurophysiology ◽

10.1152/jn.1997.78.5.2235 ◽

1997 ◽

Vol 78 (5) ◽

pp. 2235-2245 ◽

Cited By ~ 27

Author(s):

Xiao Wen Fu ◽

Borys L. Brezden ◽

Shu Hui Wu

Keyword(s):

Membrane Potential ◽

Input Resistance ◽

Resting Potential ◽

Extracellular Potassium ◽

Current Clamp ◽

Lateral Lemniscus ◽

Inward Current ◽

Dorsal Nucleus ◽

Maximal Activation

Fu, Xiao Wen, Borys L. Brezden, and Shu Hui Wu. Hyperpolarization-activated inward current in neurons of the rat's dorsal nucleus of the lateral lemniscus in vitro. J. Neurophysiol. 78: 2235–2245, 1997. The hyperpolarization-activated current ( I h) underlying inward rectification in neurons of the rat's dorsal nucleus of the lateral lemniscus (DNLL) was investigated using whole cell patch-clamp techniques. Patch recordings were made from DNLL neurons of young rats (21–30 days old) in 400 μm tissue slices. Under current clamp, injection of negative current produced a graded hyperpolarization of the cell membrane, often with a gradual sag in the membrane potential toward the resting value. The rate and magnitude of the sag depended on the amount of hyperpolarizing current. Larger current resulted in a larger and faster decay of the voltage. Under voltage clamp, hyperpolarizing voltage steps elicited a slowly activating inward current that was presumably responsible for the sag observed in the voltage response to a steady hyperpolarizing current recorded under current clamp. Activation of the inward current ( I h) was voltage and time dependent. The current just was seen at a membrane potential of −70 mV and was activated fully at −140 mV. The voltage value of half-maximal activation of I h was −78.0 ± 6.0 (SE) mV. The rate of I h activation was best approximated by a single exponential function with a time constant that was voltage dependent, ranging from 276 ± 27 ms at −100 mV to 186 ± 11 ms at −140 mV. Reversal potential ( E h) of I h current was more positive than the resting potential. Raising the extracellular potassium concentration shifted E h to a more depolarized value, whereas lowering the extracellular sodium concentration shifted E h in a more negative direction. I h was sensitive to extracellular cesium but relatively insensitive to extracellular barium. The current amplitude near maximal-activation (about −140 mV) was reduced to 40% of control by 1 mM cesium but was reduced to only 71% of control by 2 mM barium. When the membrane potential was near the resting potential (about −60 mV), cesium had no effect on the membrane potential, current-evoked firing rate and input resistance but reduced the spontaneous firing. When the membrane potential was more negative than −70 mV, cesium hyperpolarized the cell, decreased current-evoked firing and increased the input resistance. I h in DNLL neurons does not contribute to the normal resting potential but may enhance the extent of excitation, thereby making the DNLL a consistently powerful inhibitory source to upper levels of the auditory system.

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Performance changes of venous valves following tissue treatment with novel in vitro system

Phlebology The Journal of Venous Disease ◽

10.1177/0268355518804360 ◽

2018 ◽

Vol 34 (5) ◽

pp. 347-354

Author(s):

Garrett Easson ◽

Megan Laughlin ◽

Hanna Jensen ◽

Kevin Haney ◽

Marc Girardot ◽

...

Keyword(s):

High Speed ◽

In Vitro Model ◽

Closing Time ◽

Venous Valve ◽

Orifice Area ◽

Treatment Methods ◽

Venous Valves ◽

Significant Difference ◽

Vitro Model

Objectives The purpose of this study is to test venous valve performance and identify differences between native tissue and replacement devices developed with traditional tissue treatment methods using a new in vitro model with synchronized hemodynamic parameters and high-speed valve image acquisition. Methods An in vitro model mimicking the venous circulation to test valve performance was developed using hydrostatic pressure driven flow. Fresh and glutaraldehyde-treated vein segments were placed in the setup and opening/closing of the valves was captured by a high-speed camera. Hemodynamic data were obtained using synchronized hardware and virtual instrumentation. Results Geometric orifice area and opening/closing time of the valves was evaluated at the same hemodynamic conditions. A reduction in geometric orifice area of 27.2 ± 14.8% (p < 0.05) was observed following glutaraldehyde fixation. No significant difference in opening/closing time following chemical fixation was observed. Conclusions The developed in vitro model was shown to be an effective method for measuring the performance of venous valves. The observed decrease in geometric orifice area following glutaraldehyde treatment indicates a decrease in flow through the valve, demonstrating the consequences of traditional tissue treatment methods.

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