Ruthenium Red Research Articles

7-Hydroxy Frullanolide, a sesquiterpene lactone, increases intracellular calcium amounts, lowers CD4+ T cell and macrophage responses, and ameliorates DSS-induced colitis

Sesquiterpene lactones are a class of anti-inflammatory molecules obtained from plants belonging to the Asteraceae family. In this study, the effects of 7-hydroxy frullanolide (7HF), a sesquiterpene lactone, in inhibiting CD4+ T cell and peritoneal macrophage responses were investigated. 7HF, in a dose dependent manner, lowers CD69 upregulation, IL2 production and CD4+ T cell cycling upon activation with the combination of anti-CD3 and anti-CD28. Further mechanistic studies demonstrated that 7HF, at early time points, increases intracellular Ca2+ amounts, over and above the levels induced upon activation. The functional relevance of 7HF-induced Ca2+ increase was confirmed using sub-optimal amounts of BAPTA, an intracellular Ca2+ chelator, which lowers lactate and rescues CD4+ T cell cycling. In addition, 7HF lowers T cell cycling with the combination of PMA and Ionomycin. However, 7HF increases CD4+ T cell cycling with sub-optimal activating signals: only PMA or anti-CD3. Furthermore, LPS-induced nitrite and IL6 production by peritoneal macrophages is inhibited by 7HF in a Ca2+-dependent manner. Studies with Ca2+ channel inhibitors, Ruthenium Red and 2-Aminoethoxydiphenyl borate, lowers the inhibitory effects of 7HF on CD4+ T cell and macrophage responses. In silico studies demonstrated that 7HF binds to Ca2+ channels, TRPV1, IP3R and SERCA, which is mechanistically important. Finally, intraperitoneal administration of 7HF lowers serum inflammatory cytokines, IFNγ and IL6, and reduces the effects of DSS-induced colitis with respect to colon length and colon damage. Overall, this study sheds mechanistic light on the anti-inflammatory potential of 7HF, a natural plant compound, in lowering immune responses.

Mechanotransduction channel Piezo is widely expressed in the spider, Cupiennius salei, mechanosensory neurons and central nervous system

Mechanosensory neurons use mechanotransduction (MET) ion channels to detect mechanical forces and displacements. Proteins that function as MET channels have appeared multiple times during evolution and occur in at least four different families: the DEG/ENaC and TRP channels, as well as the TMC and Piezo proteins. We found twelve putative members of MET channel families in two spider transcriptomes, but detected only one, the Piezo protein, by in situ hybridization in their mechanosensory neurons. In contrast, probes for orthologs of TRP, ENaC or TMC genes that code MET channels in other species did not produce any signals in these cells. An antibody against C. salei Piezo detected the protein in all parts of their mechanosensory cells and in many neurons of the CNS. Unspecific blockers of MET channels, Ruthenium Red and GsMTx4, had no effect on the mechanically activated currents of the mechanosensory VS-3 neurons, but the latter toxin reduced action potential firing when these cells were stimulated electrically. The Piezo protein is expressed throughout the spider nervous system including the mechanosensory neurons. It is possible that it contributes to mechanosensory transduction in spider mechanosensilla, but it must have other functions in peripheral and central neurons.

Concurrent validity of biofilm detection by wound blotting on hard-to-heal wounds.

Wound biofilms delay healing of hard-to-heal wounds. Convenient biofilm identification tools for clinical settings are currently not available, hindering biofilm-based wound management. Wound blotting with biofilm staining is a potential tool for biofilm detection, owing to its convenience. Although predictive validity of wound blotting has been established, it is necessary to confirm its concurrent validity. Furthermore, current staining systems employing ruthenium red have some disadvantages for clinical use. This study aimed to evaluate the usability of alcian blue as a substitute for ruthenium red. Both in vitro and in vivo clinical samples were used to investigate validity and usability. The in vitro study showed that proteins and extracellular DNA in biofilms did not affect staining ability of ruthenium red and alcian blue in the detection of biofilms. In the in vivo study, using a wound biofilm model with Pseudomonas aeruginosa, the staining sensitivity of ruthenium red was 88.9% and 100% for alcian blue, with correlation coefficients of signal intensities with native polyacrylamide gel electrophoresis (PAGE) of r=0.67 (p=0.035) and r=0.67 (p=0.036) for ruthenium red and alcian blue, respectively. Results from clinical samples were r=0.75 (p=0.001) for ruthenium red and r=0.77 (p<0.001) for alcian blue. The sensitivities of wound blotting staining by ruthenium red and alcian blue were very high and had a good correlation with native PAGE analysis. Because the alcian blue procedure is more convenient than the ruthenium red procedure, wound blotting with alcian blue staining would be a promising tool to guide clinicians in delivering biofilm-based wound management.

Effects of various calcium transporters on mitochondrial Ca2+ changes and oocyte maturation.

Ca2+ participates in many important cellular processes, but the underlying mechanisms are still poorly understood, especially during oocyte maturation. First, we confirmed that calcium in the culture medium was essential for oocyte maturation. Next, various inhibitors of Ca2+ channels were applied to investigate their roles in mitochondrial Ca2+ changes and oocyte maturation. Our results showed that Trmp7, Orai, T-type Ca2+ channels and Na+ /Ca2+ exchanger complex (NCLX) were important for oocyte maturation. Trmp7 inhibition delayed germinal vesicle breakdown. Orai and NCLX inhibition significantly weakened the distribution of mitochondrial Ca2+ around the nucleus compared to the Ctrl group. Interestingly, even T-type Ca2+ channels-specific inhibitor Mibefradil blocked germinal vesicle breakdown; mitochondrial Ca2+ surrounding the nucleus still was maintained at a high level without spindle formation. Two calcium transporter inhibitors, Thapsigargin and Ruthenium Red, which have been confirmed to inhibit oocyte activation, did not significantly affect oocyte maturation. Increasing the knowledge of calcium transport may provide a basis to build on for improving oocyte in vitro maturation in human assisted reproduction clinics.

Activation of TRPV4 channels leads to a consistent tocolytic effect on human myometrial tissues.

Human myometrium is a therapeutic target for labor induction and preterm labor. This study aimed to assess the physiological role of alternative calcium conductance on contractions triggered by uterotonic drugs in human myometrium. Membrane conductances, supported by TRPV channels, may provide alternative pathways to control either free intracellular and/or submembrane Ca2+-concentration, which in turn will modulate membrane polarization and contractile responses. Uterine biopsies were obtained from consenting women undergoing elective caesarean delivery at term without labor (N = 22). Isometric tension measurements were performed on uterine smooth muscle strips (n = 132). Amplitude, frequency, and area under the curve (AUC) of phasic contractions, as well as resting tone, were measured under various experimental conditions. Immuno histo- and cyto-chemistry, as well as Western blot analyses, have been performed with specific antibodies against TRPV1, TRPV3, and TRPV4 proteins. TRPV4 agonists; GSK1016790A, 4αPDD, and 5,6-EET were used to assess the role of TRPV4 channels on rhythmic activity triggered by 30-300 nM oxytocin. 5 μM of ruthenium red was used as an efficient blocker of ionic current through TRPV4 channels. Nanomolar concentrations of iberiotoxin (IbTX) were also used to confirm the downstream involvement of BKCa channels in controlling uterine reactivity and contractility. The expression of TRPV3 and TRPV4 isoforms has now been demonstrated in human myometrial tissue and cell culture. Nanomolar concentrations of the TRPV4 agonists, (either GSK1016790A or 4αPDD) abolished the rhythmic contractions, resulting in a rapid and consistent tocolytic effect. While 5 μM of ruthenium reversed this tocolytic effect. The addition of IbTX (a BKCa channel blocker) reversed the effects of GSK1016790A. Carvacrol, a TRPV3 agonist, had similar tocolytic effects on rhythmic contractions albeit at higher concentrations. This inhibitory effect was also reversed by ruthenium red. Collectively, these data suggest that activation of TRPV4 leads to a Ca2+ entry and subsequent BKCa channel activation (increase in open state probability), which in turn hyperpolarizes the myometrial cell membrane, inactivating L-type Ca2+ channels and efficiently abrogates contractile activity. Consequently, alternative Ca2+ conductance supported by TRPV4 plays a physiological role in the modulation of myometrial reactivity.

Structure Basis and Gating Mechanism of Calcium Homeostasis Modulator 2 Channel

By mediating the purinergic signaling pathway, ATP release channels play a fundamental role in many neurological functions, including the modulation of excitatory synaptic strength, long-term synaptic potentiation, and neuronal excitability. The calcium homeostasis modulators are key ATP release channels consisting of three members, CALHM1, 2 and 3. CALHM channels plays essential roles in taste and neuron transmission, and their dysregulation has been associated with various neurological disorders including Alzheimer disease and ischemic brain damage. In the absence of Ca2+, the CALHMs are activated in a voltage-dependent manner, and the current can be inhibited by small molecules such as ruthenium red (RUR). The topology of CALHMs consists of four transmembrane domains (S1-S4) and a long cytosolic domain. Despite the importance of CALHM channels, their structures and functional properties are largely unexplored.We determined cryo-EM structures of full-length human CALHM2 in the Ca2+-free open state as both hemichannel and gap junction, and RUR-bound inhibited state at resolutions up to 2.7 Å. CALHM2 forms an unpreceded undecamer. We identified a RUR binding site located underneath the S1, where a mutation of a key residue (E37R) abolished RUR inhibition by patch-clamp electrophysiology. The primary determinants for the channel gate of CALHM2 are S1 and N-terminal helix (NTH), where remarkable conformational changes are observed when comparing the two functional states. The Ca2+-free hemichannel favors S1 in a vertical conformation, resulting in a large open pore. In the RUR-bound state, the RUR supports S1 in a lifted conformation, thus occluding the ion conducting pore. We propose a two-stage mechanism, whereby S1 adjusts the pore size coarsely and the NTH makes fine adjustments, eventually physically occluding the pore. Our work elucidates the molecular principles, overall architectures, stoichiometry of CALHM2 channel and its gating mechanism.

Microscopy Methods for Biofilm Imaging: Focus on SEM and VP-SEM Pros and Cons.

Simple SummaryBacterial biofilms cause infections that are often resistant to antibiotic treatments. Research about the formation and elimination of biofilms cannot be undertaken without detailed imaging techniques. In this review, traditional and cutting-edge microscopy methods to study biofilm structure, ultrastructure, and 3-D architecture, with particular emphasis on conventional scanning electron microscopy and variable pressure scanning electron microscopy, are addressed, with the respective advantages and disadvantages. When ultrastructural characterization of biofilm matrix and its embedded bacterial cells is needed, as in studies on the effects of drug treatments on biofilm, scanning electron microscopy with customized protocols such as the osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA), and ionic liquid (IL) must be preferred over other methods for the following: unparalleled image quality, magnification and resolution, minimal sample loss, and actual sample structure preservation. The first step to make a morphological assessment of the effect of the various pharmacological treatments on clinical biofilms is the production of images that faithfully reflect the structure of the sample. The extraction of quantitative parameters from images, possible using specific software, will allow for the scanning electron microscopy morphological evaluation to no longer be considered as an accessory technique, but a quantitative method to all effects.Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure. This review illustrates the most widely used microscopy techniques in biofilm investigations, focusing on traditional and innovative scanning electron microscopy techniques such as scanning electron microscopy (SEM), variable pressure SEM (VP-SEM), environmental SEM (ESEM), and the more recent ambiental SEM (ASEM), ending with the cutting edge Cryo-SEM and focused ion beam SEM (FIB SEM), highlighting the pros and cons of several methods with particular emphasis on conventional SEM and VP-SEM. As each technique has its own advantages and disadvantages, the choice of the most appropriate method must be done carefully, based on the specific aim of the study. The evaluation of the drug effects on biofilm requires imaging methods that show the most detailed ultrastructural features of the biofilm. In this kind of research, the use of scanning electron microscopy with customized protocols such as osmium tetroxide (OsO4), ruthenium red (RR), tannic acid (TA) staining, and ionic liquid (IL) treatment is unrivalled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preservation. The combined use of innovative SEM protocols and 3-D image analysis software will allow for quantitative data from SEM images to be extracted; in this way, data from images of samples that have undergone different antibiofilm treatments can be compared.

Ruthenium red: a highly efficient and versatile cell staining agent for single-cell analysis using inductively coupled plasma time-of-flight mass spectrometry.

Staining of biological cells with heavy metals can increase their visibility in mass spectrometry. In this study, the potential of ruthenium red (RR) as a staining agent for single-cell analysis by inductively coupled plasma time-of-flight mass spectrometry (SC-ICP-TOF-MS) is explored using two different yeast strains and one algal species. Time-of-flight mass spectrometry allows the simultaneous detection of Ru and multiple intrinsic elements in single cells. Ru has a better correlation with Mg than with P in Saccharomyces cerevisiae (S. cerevisiae) cells. For the three tested strains, the staining efficiency of RR exceeded 96%; the staining strengths were 30-32 ag μm-2 for the yeast cells and 59 ag μm-2 for the algal cells. By deriving the cell volume of single cells from their Ru mass, the concentration of Mg and P in individual cells of S. cerevisiae can be calculated. Elemental concentrations of Mg and P were highly variable in the cell individuals, with their 25-75 percentile values of 0.10-0.19 and 0.76-2.07 fg μm-3, respectively. RR staining has several advantages: it is fast, does not affect cell viability and is highly efficient. Provided that the shape of the individual cells of a culture is similar, Ru staining allows the elemental content to be directly correlated with the cell volume to accurately calculate the intracellular concentration of target elements in single cells. Therefore, RR can be a promising cell staining agent for future application in SC-ICP-TOF-MS research.

Allyl isothiocyanate (AITC) activates nonselective cation currents in human cardiac fibroblasts: possible involvement of TRPA1

The effects of allyl isothiocyanate (AITC), transient receptor potential ankyrin 1 (TRPA1) agonist, on cultured human cardiac fibroblasts were examined by measuring intracellular Ca2+ concentration [Ca2+]i and whole-cell voltage clamp techniques. AITC (200 μM) increased Ca2+ entry in the presence of [Ca2+]i. Ruthenium red (RR) (30 μM), and La3+ (0.5 mM), a general cation channel blocker, inhibited AITC-induced Ca2+ entry. Under the patch pipette filled with Cs+- and EGTA-solution, AITC induced the current of a reversal potential (Er) of approximately +0 mV. When extracellular Na+ ion was changed by NMDG+, the inward current activated by AITC was markedly reduced. La3+ and RR inhibited the AITC-induced current. The conventional RT-PCR analysis, Western blot, and immunocytochemical studies showed TRPA1 mRNA and protein expression. The present study shows the first evidence for functional Ca2+-permeable nonselective cation currents induced by AITC, possibly via TRPA1 in human cardiac fibroblast.

NADPH Oxidase-derived ROS promote mitochondrial alkalization under salt stress in Arabidopsis root cells

ABSTRACT The plasma membrane NADPH Oxidase-derived ROS as signaling molecules play crucial roles in salt stress response. As the motor organelle of cells, mitochondria are also important for salt tolerance. However, the possible interaction between NADPH Oxidase-derived ROS and mitochondria is not well studied. Here, a transgenic Arabidopsis expressing mitochondrial matrix-targeted pH-sensitive indicator cpYFP was used to monitor the pH dynamics in root cells under salt stress. A significant alkalization in mitochondria was observed when the root was exposed to NaCl or KCl, but not osmotic stress such as isotonic mannitol. Interestingly, when pretreated with the NADPH Oxidase inhibitor DPI, the mitochondrial alkalization in root cells was largely abolished. Genetic evidence further showed that salt-induced mitochondrial alkalization was significantly reduced in the loss of function mutant atrbohF . Pretreatment with endocytosis-related inhibitor PAO or TyrA23, which inhibited the ROS accumulation under salt treatment, almost abolished this effect. Furthermore, [Ca2+]cyt increase might also play important roles by affecting ROS generation to mediate salt-induced mitochondrial alkalization as indicated by treatment with plasma membrane Ca2+ channel inhibitor LaCl3 and mitochondrial Ca2+ uniporter inhibitor Ruthenium Red. Together, these results suggest that the plasma membrane NADPH Oxidase-derived ROS promote the mitochondrial alkalization under salt treatment, providing a possible link between different cellular compartments under salt stress.

Piezo1 channels mediate trabecular meshwork mechanotransduction and promote aqueous fluid outflow.

Trabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humour drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humour outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure. Its activation resulted in intracellular signals that altered organization of the cytoskeleton and cell-extracellular matrix contacts and modulated the trabecular component of aqueous outflow whereas another channel, TRPV4, mediated a delayed mechanoresponse. This study helps elucidate basic mechanotransduction properties that may contribute to intraocular pressure regulation in the vertebrate eye. Chronic elevations in intraocular pressure (IOP) can cause blindness by compromising the function of trabecular meshwork (TM) cells in the anterior eye, but how these cells sense and transduce pressure stimuli is poorly understood. Here, we demonstrate functional expression of two mechanically activated channels in human TM cells. Pressure-induced cell stretch evoked a rapid increase in transmembrane current that was inhibited by antagonists of the mechanogated channel Piezo1, Ruthenium Red and GsMTx4, and attenuated in Piezo1-deficient cells. The majority of TM cells exhibited a delayed stretch-activated current that was mediated independently of Piezo1 by TRPV4 (transient receptor potential cation channel, subfamily V, member 4) channels. Piezo1 functions as the principal TM transducer of physiological levels of shear stress, with both shear and the Piezo1 agonist Yoda1 increasing the number of focal cell-matrix contacts. Analysis of TM-dependent fluid drainage from the anterior eye showed significant inhibition by GsMTx4. Collectively, these results suggest that TM mechanosensitivity utilizes kinetically, regulatory and functionally distinct pressure transducers to inform the cells about force-sensing contexts. Piezo1-dependent control of shear flow sensing, calcium homeostasis, cytoskeletal dynamics and pressure-dependent outflow suggests potential for a novel therapeutic target in treating glaucoma.

Ruthenium red attenuates brown adipose tissue thermogenesis in rats.

Ruthenium red (RR) is a non-selective antagonist of the temperature-sensitive Transient Receptor Potential (TRP) channels and it is an important pharmacological tool in thermoregulatory research. However, the effect of RR on thermoeffector activity is not well established. Here we evaluated the effect of RR on cold-defense thermoeffectors induced by menthol, an agonist of the cold-sensitive TRPM8 channel. Adult male Wistar rats were used. Epidermal treatment with menthol raised deep body temperature due to an increase in oxygen consumption (an index of thermogenesis), a reduction in heat loss index (an index of cutaneous vasoconstriction), and an induction in warmth-seeking behavior in a two-temperature choice apparatus. Pretreatment with RR attenuated the menthol-induced increase in deep body temperature and oxygen consumption, but it did not affect heat loss index and warmth-seeking behavior. To stimulate brown adipose tissue thermogenesis, rats were treated with CL 316,243, a potent and selective β3-adrenoceptor agonist. CL 316,243 increased deep body temperature, which was attenuated by RR pretreatment. We conclude that RR reduces brown adipose tissue thermogenesis induced by menthol and CL 316,243, independent of effects at the thermal sensor level (i.e., TRPM8).

Suppression of ventricular arrhythmia by mitochondrial calcium uptake via mitochondrial calcium uniporter in the ischemic heart failure mice

Abstract Background In heart failure (HF), diastolic calcium (Ca) leak from sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) causes delayed after depolarization (DAD), leading ventricular arrhythmias (VAs). Recent study reported that Ca uptake into mitochondria via mitochondrial calcium uniporter (MCU) suppress Ca waves (CaWs) and DAD in catecholaminergic polymorphic ventricular tachycardia, in which diastolic Ca leak is thought to be a major cause of VAs as in HF. However, such anti-arrhythmic effect of mitochondrial Ca uptake via MCU remains unclear in HF. Purpose We sought to investigate whether mitochondrial Ca uptake via MCU decreases CAWs and VAs incidence in ischemic HF mice. Methods Ten-week-old male C57BL/6J mice were divided into 2 groups; sham operation mice (Sham) or HF mice (HF) in which myocardial infarction was induced by left coronary artery ligation. After 4–6 weeks, cardiomyocyte or mitochondria was isolated respectively from the myocardium of Sham and the non-infarct myocardium of HF. Influence of MCU activation on Ca dynamics, VA inducibility and left ventricular hemodynamics were evaluated using Kaemenpferol, a MCU activator. Intracellular Ca dynamics and mitochondrial Ca uptake were measured in isolated cardiomyocytes loaded with Fluo-4 AM on an epifluorescence microscopy and by estimating the extra-mitochondrial Ca reduction with Fluo-5N on a spectrofluoro-photometer, respectively. VAs was induced by programmed stimulation in the Langendorff perfused hearts. Left ventricular (LV) pressure was measured using a microtip transducer catheter. Finally, the effect of intravenous administration of Kaempferol (5mg/kg) on hemodynamic parameters was examined 30 minutes after administration in Sham and HF. Results HF mice showed left ventricular dysfunction, as well as the increased heart and lung weights compared to Sham. MCU protein expression in cardiomyocytes did not differ between Sham and HF. Kaempferol increased mitochondrial Ca uptake in the isolated mitochondria both in Sham and HF. The number of the diastolic CaWs was higher in HF compared to Sham. Such increased number of CaWs in HF was attenuated by 10 μM Kaempferol, which was, however, abolished by a MCU blocker Ruthenium Red. The incidence of induced VA was significantly higher in HF than Sham, which was suppressed by Kaempferol. In vivo measurements, intravenous administration of Kaempferol did not show significant changes in hemodynamic parameters in Sham and HF mice. Conclusions Mitochondrial Ca uptake via MCU suppresses CaWs and VAs, but did not change LV hemodynamics in HF. Whereas traditional antiarrhythmic drugs have limited use in heart failure patients, a novel strategy that promotes Ca uptake into mitochondria might be a new and safer option for treating VAs in HF. Funding Acknowledgement Type of funding source: None

Oligogalacturonide-accelerated healing of mechanical wounding in tomato fruit requires calcium-dependent systemic acquired resistance

Mechanical wounding causes significant economic losses of fresh produce due to accelerated senescence and spoilage as well as loss of nutritional value. Here, pre-application of oligogalacturonides (OGs) enzymatically hydrolyzed from apple pectin effectively reduced the healing times of mechanical wounds from>24 h in mock groups to 12 h, and the Botrytis cinerea infection rate was reduced from 37.5% to 12.5%. OGs accordingly increased callose deposition; SlPR1, SlPAL and SlHCT gene expression; and phenylalanine ammonia-lyase (PAL) activity around the wounds. Inhibition of Ca2+ signaling using the inhibitor Ruthenium Red markedly inhibited OG accelerated healing of mechanical wounding on fruit. SlPG2, SlEXP1, and SlCEL2 mRNAs accumulation was reduced in OG-elicited tomato fruit compared to water-treated fruit with subsequent retardation of the fruit softening during ripening. These results indicated that apple pectin OGs accelerate wound healing and inhibit fruit softening by activating calcium signaling in tomato fruits during postharvest storage.

Ruthenium red, mitochondrial calcium uniporter inhibitor, attenuates cognitive deficits in STZ-ICV challenged experimental animals

Alzheimer's disease (AD) is a progressive neurodegenerative disorder with cardinal features of cognitive dysfunction in an individual. Recently, the blockade of mitochondrial calcium uniporter (MCU) exhibits neuroprotective activity in experimental animals. However, the therapeutic potential of MCU has not yet been established in the management of AD. Therefore, the present study explored the therapeutic potential of either Ruthenium red (RR), a MCU blocker, or Spermine, a MCU opener, on the extent of mitochondrial calcium accumulation, function, integrity and bioenergetics in hippocampus, pre-frontal cortex and amygdale of ICV-STZ challenged rats. Experimental AD was induced in male rats by intracerebroventricular injection of streptozotocin (ICV-STZ) on day-1 (D-1) of the experimental protocol at a sub-diabetogenic dose (3 mg/kg) twice at an interval of 48 h into both rat lateral ventricles. RR attenuated ICV-STZ-induced memory-related behavioral abnormalities in Morris water maze and Y-maze tests. RR also attenuated ICV-STZ-induced decrease in the level of acetylcholine and activity of choline acetyltransferase and, increase in the activity of acetylcholinestarase in memory-sensitive rat brain regions. Further, RR attenuated mitochondrial toxicity in terms of reducing mitochondrial calcium accumulation and improving the mitochondrial function, integrity and bioenergetics in memory-sensitive brain regions of ICV-STZ challenged rats. Furthermore, RR attenuated the percentage of apoptotic cells in ICV-STZ challenged rat brain regions. However, Spermine did not alter ICV-STZ-induced behavioral, biochemical and molecular observations in any of the brain regions. These observations indicate the fact that the MCU blockage could be a potential therapeutic option in the management of sporadic type of AD.

Targeting the Proline-Rich Tyrosine Kinase 2 /Mitochondrial Calcium Uniporter Pathway, Ruthenium Red, and Nimodipine alone and in Combination Evoked Neuroprotective Effect in Experimentally Induced Brain Stroke in Rats

Targeting the Proline-Rich Tyrosine Kinase 2 /Mitochondrial Calcium Uniporter Pathway, Ruthenium Red, and Nimodipine alone and in Combination Evoked Neuroprotective Effect in Experimentally Induced Brain Stroke in Rats

Responses to transient receptor potential (TRP) channel agonists inChlamydomonas reinhardtii

ABSTRACTPungent substances, such as capsaicin and gingerol, activate the transient receptor potential (TRP)-V1 channel and affect the feeding behaviors of animals. To gain insight into how living organisms have acquired a sense for pungent substances, we explored the response to TRP agonists in a protist, Chlamydomonas reinhardtii. When capsaicin or gingerol was applied to wild-type cells, they became immotile, with flagella detaching from the cell body. The degree of deflagellation was nearly halved in a mutant defective in the TRP channel ADF1. Deflagellation in the adf1 mutant was inhibited further by Ruthenium Red, indicating ADF1 and another TRP channel are involved in the deflagellation response. The response to capsaicin and gingerol was not inhibited by TRPV1-specific blockers such as 4-(3-Chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide (BCTC) and capsazepine. When capsaicin or gingerol was applied to wild-type cells in the presence of Ruthenium Red, a large proportion lost motility while flagella remained attached, suggesting that flagella stop contributing to motility, at least in part, through a TRP-channel-independent pathway. These results indicate that pungent compounds such as capsaicin and gingerol induce loss of flagellar motility and flagellar detachment in C. reinhardtii cells.

Paper-based colorimetric sensor for easy and simple detection of polygalacturonase activity aiming for diagnosis of Allium white rot disease

Polygalacturonase (PG) activity in plants can serve as an important index for plant disease. However, the conventional method to detect PG activity is a complex process and requires a skilled technician and expensive analytical equipment. In this study, a paper-based colorimetric sensor was developed based on the principle of the ruthenium red (RR) dye method for easy and simple measurement of PG activity. The proposed paper-based sensor has a three-layer structure for detection of PG activity in samples. The sensor sensitivity was enhanced by optimizing the pH of the sodium acetate buffer used in polygalacturonic acid (PGA)-RR complex formation and the reaction temperature for PG and the PGA-RR complex. Further, for quantitative analysis of PG activity, Delta RGB analysis was conducted to detect color changes in the sensing window of the sensor. Results presented that the linear measurement range of the paper sensor was 0.02–0.1 unit with the limit of detection of 0.02 unit, which showed a similar detection range, but a lower detection limit, compared to the spectrophotometry. Furthermore, PG activity based on culture condition was measured using samples from Sclerotium cepivorum to verify the potential application of the developed paper-based sensor in the field. The measured activity showed no statistically significant difference from the values obtained from the spectrophotometry at 95% confidence level. Therefore, the paper-based colorimetric sensor can be used to predict plant diseases in Allium crops during the stage of pathogen invasion, potentially contributing to the improvement of crop production.

Polynuclear Ruthenium Amines Inhibit K2P Channels via a “Finger in the Dam” Mechanism

The trinuclear ruthenium amine ruthenium red (RuR) inhibits diverse ion channels, including K2P potassium channels, TRPs, the calcium uniporter, CALHMs, ryanodine receptors, and Piezos. Despite this extraordinary array, there is limited information for how RuR engages targets. Here, using X-ray crystallographic and electrophysiological studies of an RuR-sensitive K2P, K2P2.1 (TREK-1) I110D, we show that RuR acts by binding an acidic residue pair comprising the "Keystone inhibitor site" under the K2P CAP domain archway above the channel pore. We further establish that Ru360, a dinuclear ruthenium amine not known to affect K2Ps, inhibits RuR-sensitive K2Ps using the same mechanism. Structural knowledge enabled a generalizable design strategy for creating K2P RuR "super-responders" having nanomolar sensitivity. Together, the data define a "finger in the dam" inhibition mechanism acting at a novel K2P inhibitor binding site. These findings highlight the polysite nature of K2P pharmacology and provide a new framework for K2P inhibitor development.

Analysis of Differential Gene Expression in Response to Anisotropic Stretch using a Systems Model of Cardiac Myocyte Mechanotransduction

Cardiomyocytes transduce various mechanical signals into transcriptional responses associated with ventricular hypertrophy and remodeling. The organization of the sarcomere and cytoskeleton suggest that myocytes may respond differentially to mechanical loading applied either parallel or transverse to the long axis of the myofiber. The regulatory networks underlying these differential responses, however, remain unknown. Micropatterned mouse neonatal cardiomyocytes were stretched biaxially, the major axis of strain being either parallel or transverse to the myofiber orientation, to examine the differential effects of anisotropic stretch on gene expression. At 30 minutes and 4 hours of stretch, RNA sequencing identified 843 differentially expressed genes of which 830 were regulated by longitudinal stretch and 82 regulated by transverse stretch. An ordinary differential equation model of cardiac myocyte mechanosignaling predicted the expression of 784 genes with an accuracy of 72%. By simulating inhibition of mechanoreceptors, the model predicted the greatest inhibition of genes induced by transverse stretch when angiotensin II (AngII) and endothelin-1 (ET-1) receptors were blocked. These predictions were confirmed by experiments which showed a significantly greater inhibition (48%) in the transverse-stretch induced expression of Ctgf, Fosl2, Mafk and Nuak1 by combined inhibition of AngII and ET1 receptors with 1uM Losartan and 100uM/10uM BQ123/BQ788, respectively, compared with combined inhibition of the L-type Ca channel (5uM Nifedipine), sodium hydrogen exchanger (10uM HOE642) and TRP channels (1uM Ruthenium Red) which resulted in 18% inhibition. A graph node centrality analysis showed that 22 signaling molecules primarily in MAPK pathways are the dominant regulators of the expression of 20 genes in response to transverse stretch. Thus, we conclude that transverse stretch induces differential gene responses by stimulating AngII and ET1 receptors and activating 9 transcriptional regulators via the MAPK pathways.