Changes In Membrane Potential Research Articles

An α-π transition in S6 shapes the conformational cycle of the bacterial sodium channel NavAb.

Voltage-gated sodium channels play an important role in electrical signaling in excitable cells. In response to changes in membrane potential, they cycle between nonconducting and conducting conformations. With recent advances in structural biology, structures of sodium channels have been captured in several distinct conformations, which are thought to represent different functional states. However, it has been difficult to capture the intrinsically transient open state. We recently showed that a proposed open state of the bacterial sodium channel NavMs was not conductive and that a conformational change involving a transition to a π-helix in the pore-lining S6 helix converted this structure into a conducting state. However, the relevance of this structural feature in other sodium channels, and its implications for the broader gating cycle, remained unclear. Here, we propose a comparable open state of another class of bacterial channel from Aliarcobacter butzleri (NavAb) with characteristic pore hydration, ion permeation, and drug binding properties. Furthermore, we show that a π-helix transition can lead to pore opening and that such a conformational change blocks fenestrations in the inner helix bundle. We also discover that a region in the C-terminal domain can undergo a disordering transition proposed to be important for pore opening. These results support a role for a π-helix transition in the opening of NavAb, enabling new proposals for the structural annotation and drug modulation mechanisms in this important sodium channel model.

Synthesis, Antiproliferative Evaluation and QSAR Analysis of Novel Halogen- and Amidino-Substituted Benzothiazoles and Benzimidazoles.

Syntheses of 6-halogen-substituted benzothiazoles were performed by condensation of 4-hydroxybenzaldehydes and 2-aminotiophenoles and subsequent O-alkylation with appropriate halides, whereas 6-amidino-substituted benzothiazoles were synthesized by condensation of 5-amidino-2-aminothiophenoles and corresponding benzaldehydes. While most of the compounds from non-substituted and halogen-substituted benzothiazole series showed marginal antiproliferative activity on tested tumor cell lines, amidino benzazoles exhibited stronger inhibitory activity. Generally, imidazolyl benzothiazoles showed pronounced and nonselective activity, with the exception of 36c which had a strong inhibitory effect on HuT78 cells (IC50 = 1.6 µM) without adverse cytotoxicity on normal BJ cells (IC50 >100 µM). Compared to benzothiazoles, benzimidazole structural analogs 45a-45c and 46c containing the 1,2,3-triazole ring exhibited pronounced and selective antiproliferative activity against HuT78 cells with IC50 < 10 µM. Moreover, compounds 45c and 46c containing the methoxy group at the phenoxy unit were not toxic to normal BJ cells. Of all the tested compounds, benzimidazole 45a with the unsubstituted phenoxy central core showed the most pronounced cell growth inhibition on THP1 cells in the nanomolar range (IC50 = 0.8 µM; SI = 70). QSAR models of antiproliferative activity for benzazoles on T-cell lymphoma (HuT78) and non-tumor MDCK-1 cells elucidated the effects of the substituents at position 6 of benzazoles, demonstrating their dependence on the topological and spatial distribution of atomic mass, polarizability, and van der Waals volumes. A notable cell cycle perturbation with higher accumulation of cells in the G2/M phase, and a significant cell increase in subG0/G1 phase were found in HuT78 cells treated with 36c, 42c, 45a-45c and 46c. Apoptotic morphological changes, an externalization of phosphatidylserine, and changes in the mitochondrial membrane potential of treated cells were observed as well.

Conditions for Kir-induced bistability of membrane potential in capillary endothelial cells

A simplified model for electrophysiology of endothelial cells is used to examine the conditions that can lead to bistability of membrane resting potential. The model includes the effects of inward-rectifying potassium (Kir) ion channels, whose current–voltage relationship shows an interval of negative slope and whose maximum conductance is dependent on the extracellular potassium concentration. The background current resulting from other types of channels is assumed to be linearly related to membrane potential. A method is presented for identifying the boundaries in the parameter space for the background currents of the regions of bistability. It is shown that these regions are relatively narrow and depend on extracellular potassium concentration. The results are used to define conditions leading to transitions between depolarized and hyperpolarized membrane states. These behaviors can influence the properties of conducted responses, in which changes in membrane potential are propagated along blood vessel walls. Conducted responses are important in the local regulation of blood flow in the brain and other tissues.

Necroptosis signaling and NLRP3 inflammasome cross-talking in epithelium facilitate Pseudomonas aeruginosa mediated lung injury

Pseudomonas aeruginosa induced acute lung injury is such a serious risk to public health, but the pathological regulation remains unclear. Here, we reported that PA mediated epithelial necroptosis plays an important role in pathological process. Pharmacological and genomic ablation of necroptosis signaling ameliorate PA mediated ALI and pulmonary inflammation. Our results further proved NLRP3 inflammasome to involve in the process. Mechanism investigation revealed the cross-talking between inflammasome activation and necroptosis that MLKL-dependent necroptosis signaling promotes the change of mitochondrial membrane potential for the release of reactive oxygen species (ROS), which is the important trigger for functional inflammasome activation. Furthermore, antioxidants such as Mito-TEMPO was confirmed to significantly restrain inflammasome activation in epithelium, resulting in a reduction in PA induced pulmonary inflammation. Taken together, our findings revealed that necroptosis-triggered NLRP3 inflammasome in epithelium plays a crucial role in PA mediated injury, which could be a potential therapeutic target for pulmonary inflammation.

Effects of the gaseous signalling molecule nitroxyl (HNO) on myenteric neurons governing intestinal motility.

The main function of myenteric neurons is the control of gut motility. As we recently showed that nitroxyl (HNO) induces intestinal smooth muscle relaxation, it was of interest to evaluate the effects of this signalling molecule on myenteric neurons in order to distinguish its properties in regard to myocytes. Myenteric neurons isolated from the ileum of 4-10days old rats were used. HNO-induced changes in intracellular concentration of Ca2+ or membrane potential and ion currents were measured using the Ca2+-sensitive fluorescent dye fura-2 AM or by electrophysiological whole-cell recordings, respectively. Changes in intracellular thiol groups pool were evaluated using thiol tracker violet. Angeli's salt was used as HNO donor. The HNO donor Angeli's salt induced a significant increase in the cytosolic Ca2+ concentration at the concentration 50µM and a membrane hyperpolarization from a resting membrane potential of-56.1±8.0mV to-63.1±8.7mV (n=7). Although potassium channels primarily drive membrane potential changes in these cells, outwardly rectifying potassium currents were not significantly affected by 50µM Angeli's salt. Fast inward sodium currents were slightly but not significantly reduced by HNO. In more sensitive cells, HNO tended to reduce the pool of thiol groups. As in the case of smooth muscle cells, HNO causes hyperpolarization of myenteric neurons, an effect also associated with an increase in intracellular Ca2+ concentration. Pathways other than activation of potassium currents appear to drive the hyperpolarization evoked by HNO.

Fluorinated triphenylphosphonium analogs improve cell selectivity and invivo detection of mito-metformin.

Triphenylphosphonium (TPP+) conjugated compounds selectively target cancercells by exploiting their hyperpolarized mitochondrial membrane potential. Todate, studies have focused on modifying either the linker or the cargo of TPP+-conjugated compounds. Here, we investigated the biological effects ofdirect modification to TPP+ to improve the efficacy and detection of mito-metformin (MMe), a TPP+-conjugated probe we have shown to have promising preclinical efficacy against solid cancer cells. We designed, synthesized, and tested trifluoromethyl and methoxy MMe analogs (pCF3-MMe, mCF3-MMe, and pMeO-MMe) against multiple distinct human cancer cells. pCF3-MMe showed enhanced selectivity toward cancer cells compared to MMe, while retaining thesame signaling mechanism. Importantly, pCF3-MMe allowed quantitative monitoring of cellular accumulation via 19F-NMR invitro and invivo. Furthermore, adding trifluoromethyl groups to TPP+ reduced toxicity invivo while retaining anti-tumor efficacy, opening an avenue to de-risk these next-generation TPP+-conjugated compounds.

Ranolazine Effectively Ameliorated Brain Pathologies and Cognitive Decline in Rats with Trastuzumap‐Induced Chemobrain

AbstractBackgroundChemotherapy can increase survival rate of cancer patients. One of the chemotherapeutic drugs for breast cancer patients is trastuzumap (Trz). Chemotherapy not only helps to kill cancers, but it also causes several adverse effects in the patients, particularly in the brain, known as chemobrain. Chemobrain has been characterized by cognitive decline with brain mitochondrial dysfunction, hippocampal microglial activation, and loss of dendritic spines. Although several types of chemotherapy induce chemobrain, Trz‐induced chemobrain are not clearly elucidated. Ranolazine, a drug that is commonly used to treat the patients with chronic angina, shows benefits on cognitive function in doxorubicin‐induced chemobrain. However, the effects of ranolazine on brain mitochondrial function, hippocampal microglial function, hippocampal dendritic spines, and cognitive function in Trz‐induced chemobrain have never been investigated.MethodsEighteen male Wistar rats were randomly divided into two groups including control group (0.9% NSS, intraperitoneal (i.p.) injection, n=8) and Trz‐treated group (4 mg/kg for 7 days, i.p. injection, n=16). All Trz‐treated rats were randomly divided into two subgroups (n=8/ subgroup) to receive either vehicle (0.9% NSS, daily via oral gavage) or ranolazine (305 mg/kg/day, daily via oral gavage). At the end of experiment protocol, cognitive function was measured, and brains were obtained for further molecular investigation.ResultsThe results demonstrated that vehicle‐treated Trz‐rats significantly increased brain mitochondrial ROS production, increased brain mitochondrial membrane potential change, and increased brain mitochondrial swelling, suggesting brain mitochondrial dysfunction (p<0.05, Figure 1). These parameters were attenuated in ranolazine‐treated Trz‐rats (p<0.05, Figure 1). In addition, the microglial activation was found in vehicle‐treated Trz‐rats as indicated by increased the number of microglial cells, which was suppressed by the ranolazine treatment (p<0.05, Figure 1). Consistently, the dendritic spine density decreased in vehicle‐treated Trz‐rats, leading to cognitive decline (p<0.05, Figure 1). Ranolazine‐treated Trz‐rats showed an increase in dendritic spine density, resulting in the improvement of cognitive function (p<0.05, Figure 1).ConclusionsAll findings suggest that ranolazine effectively ameliorated cognitive decline in rats with Trz‐induced chemobrain. The novel findings obtained from the present study provided the therapeutic strategies which can be translated into clinical settings for better preventing chemobrain in the future.

Melatonin Improved Brain Pathologies and Cognitive Dysfunction in Rats with Trastuzumap‐Induced Chemobrain

AbstractBackgroundTrastuzumap (Trz) has been shown to significantly increase the survival rate in human epidermal growth factor receptor (HER2)‐positive breast cancer patients, whereas it also causes neuroinflammation, leading to cognitive impairment, as called “chemobrain”. The exact mechanisms of Trz‐induced chemobrain are not fully understood. Melatonin, a hormone derived from the amino acid tryptophan, has been shown to have the neuroprotection in several pathological conditions. However, the effects of melatonin on brain mitochondrial function, microglial morphology, synaptic plasticity, and cognitive function in Trz‐induced chemobrain have never been investigated.MethodEighteen male Wistar rats were randomly divided into two groups including control group (0.9% NSS, intraperitoneal (i.p.) injection, n=8) and Trz‐treated group (4 mg/kg for 7 days, i.p. injection, n=16). All Trz‐treated rats were randomly divided into two subgroups (n=8/ subgroup) to receive either vehicle (0.9% NSS, daily via oral gavage) or melatonin (10 mg/kg/day, via oral gavage). At the end of experiment protocol, cognitive function was measured, and brains were obtained for further molecular investigation.ResultTrz‐treated rats displayed significantly impaired brain mitochondrial function as indicated by increased ROS production, brain mitochondrial membrane potential change, and brain mitochondrial swelling. These parameters were attenuated in melatonin‐treated Trz‐rats (p<0.05, Figure 1). Consistently, Trz administration decreased microglial complexity, as measured by Sholl analysis, which was ameliorated by melatonin treatment (p<0.05, Figure 1). In addition, the results demonstrated that hippocampal plasticity as indicated by dendritic spine density significantly decreased in Trz‐rats, resulting in cognitive dysfunction as indicated by reducing preferent index of novel object location (NOL) and novel object recognition (NOR) test (p<0.05, Figure 1). Melatonin‐treated Trz‐rats improved hippocampal plasticity by increased dendritic spine density, leading to the improvement of cognition by increased preferent index of NOL and NOR (p<0.05, Figure 1).ConclusionThese findings suggest that melatonin effectively ameliorated cognitive dysfunction in rats with Trz‐induced chemobrain. The novel findings obtained from the present study provided the therapeutic strategies which can be translated into clinical settings for better preventing chemobrain in the future.

Buyang Huanwu decoction alleviates oxidative injury of cerebral ischemia-reperfusion through PKCε/Nrf2 signaling pathway

Ethnopharmacological relevanceIschemic stroke is a significant risk factor for human health, and Buyang Huanwu Decoction is a classical and famous Chinese formula for treating it, but without clear pharmacological mechanism. Aim of the studyThe aim of this study was to investigate that the molecular mechanism of BYHWD activation of the PKCε/Nrf2 signaling pathway to attenuate cerebral ischemia-reperfusion (I/R) oxidative damage. Materials and methodsThe MCAO method was used to establish a brain I/R injury model in SD rats, and neurological deficits were evaluated by neurological function score. Neuronal damage was observed by Nissl staining and immunofluorescence detection of MAP2 expression. Oxidative damage was observed by ROS, SOD, GSH-PX, MDA, and 8-OHdG. Changes in mitochondrial membrane potential were detected by using the fluorescent probe JC-1. The Western blot analysis detected protein expression of PKCε, P-PKCε, total Nrf2, nuclear Nrf2, HO-1, and NQO1. ResultsBYHWD significantly enhanced neural function, reduced neuronal damage, inhibited the production of ROS, decreased MDA and 8-OHdG levels, increased SOD and GSH-PX activity to reduce oxidative damage, and restored mitochondrial membrane potential. BYHWD and Nrf2 activator TBHQ increased total Nrf2, nucleus Nrf2 protein expression, and its downstream HO-1 and NQO1 proteins, and the administration of the Nrf2 inhibitor brusatol reduced the enhancing effect of BYHWD. Meanwhile, BYHWD increased the expression of PKCε and P-PKCε and the administration of the PKCε inhibitor εV1–2 reduced the effect of BYHWD in increasing the expression of PKCε, P-PKCε, nuclear Nrf2, and HO-1, as well as promoting the effect of Nrf2 translocation to the nucleus. ConclusionThis study marks the first to demonstrate that BYHWD ameliorates oxidative damage and attenuates brain I/R injury by activating the PKCε/Nrf2/HO-1 pathway.

Generation of human induced pluripotent stem cell line encoding for a genetically encoded voltage indicator Arclight A242

Genetically encoded voltage indicators (GEVIs) allow for monitoring membrane potential changes in neurons and cardiomyocytes (CMs) as an alternative to patch-clamp techniques. GEVIs facilitate non-invasive, high throughput screening of electrophysiological properties of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). A dual transgenic hiPSC line with Arclight A242 (GEVI) and an antibiotic resistance cardiac selection cassette was successfully generated from an earlier established hiPSC line MHHi001-A. After cardiac differentiation and selection, purified populations of CMs with constitutive GEVI expression can be utilized for studying cardiac development, disease modeling, and drug testing.

Neuroprotective effects of a lead compound from coral via modulation of the orphan nuclear receptor Nurr1.

To screen coral-derived compounds with neuroprotective activity and clarify the potential mechanism of lead compounds. The lead compounds with neuroprotective effects were screened by H2 O2 and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPP+ )-induced cell damage models in SH-SY5Y cells. CCK8 and LDH assays were used to detect cell viability. The anti-apoptosis of lead compounds was evaluated by flow cytometry. JC-1 and MitoSox assays were performed to examine the changes in mitochondrial membrane potential and mitochondrial ROS level. Survival of primary cortical and dopaminergic midbrain neurons was measured by MAP2 and TH immunoreactivities. The Caenorhabditis elegans (C. elegans) model was established to determine the effect of lead compounds on dopaminergic neurons and behavior changes. Three compounds (No. 63, 68, and 74), derived from marine corals, could markedly alleviate the cell damage and notably reverse the loss of worm dopaminergic neurons. Further investigation indicated that compound 63 could promote the expression of Nurr1 and inhibit neuronal apoptosis signaling pathways. Lead compounds from marine corals exerted significant neuroprotective effects， which indicated that coral might be a new and potential resource for screening and isolating novel natural compounds with neuroprotective effects. Furthermore, this study also provided a new strategy for the clinical treatment of neurodegenerative diseases such as Parkinson's disease.

Activation of TREK-1 (K2P2.1) potassium channels protects against influenza A-induced lung injury.

Influenza-A virus (IAV) infects yearly an estimated one billion people worldwide, resulting in 300,000-650,000 deaths. Preventive vaccination programs and antiviral medications represent the mainstay of therapy, but with unacceptably high morbidity and mortality rates, new targeted therapeutic approaches are urgently needed. Since inflammatory processes are commonly associated with measurable changes in the cell membrane potential (Em), we investigated whether Em hyperpolarization via TREK-1 (K2P2.1) K+ channel activation can protect against influenza-A virus (IAV)-induced pneumonia. We infected mice with IAV, which after 5 days caused 10-15% weight loss and a decrease in spontaneous activity, representing a clinically relevant infection. We then started a 3-day intratracheal treatment course with the novel TREK-1 activating compounds BL1249 or ML335. We confirmed TREK-1 activation with both compounds in untreated and IAV-infected primary human alveolar epithelial cells (HAECs) using high-throughput fluorescent imaging plate reader (FLIPR) assays. In mice, TREK-1 activation with BL1249 and ML335 counteracted IAV-induced histological lung injury and decrease in lung compliance and improved BAL fluid total protein levels, cell counts, and inflammatory IL-6, IP-10/CXCL-10, MIP-1α, and TNF-α levels. To determine whether these anti-inflammatory effects were mediated by activation of alveolar epithelial TREK-1 channels, we studied the effects of BL1249 and ML335 in IAV-infected HAEC, and found that TREK-1 activation decreased IAV-induced inflammatory IL-6, IP-10/CXCL10, and CCL-2 secretion. Dissection of TREK-1 downstream signaling pathways and construction of protein-protein interaction (PPI) networks revealed NF-κB1 and retinoic acid-inducible gene-1 (RIG-1) cascades as the most likely targets for TREK-1 protection. Therefore, TREK-1 activation may represent a novel therapeutic approach against IAV-induced lung injury.

Natural melanin-based nanoparticles with photothermal/photodynamic activities induce ovarian cancer cell death

To investigate the physicochemical characteristics of natural melanin-like nanoparticles (PDA NPs) and their synergistic anti-tumor efficacy with photothermal and photodynamic (PTT/PDT) therapy. The chemically synthesized PDA NPs were characterized using transmission electron microscope (TEM), dynamic light scattering (DLS) and Zeta potential analysis, and their photothermal and photodynamic properties were assessed with near-infrared excitation light (NIR). The antitumor efficacy of free PDA or PDA combined with NIR irradiation (0.7 and 1.0 W/cm2) was evaluated in ovarian cancer cells using flow cytometry, Cell Counting Kit-8 (CCK-8), and Transwell assay and in a mouse model bearing subcutaneous ovarian cancer xenograft. The synthesized PDA NPs showed a spherical morphology with diameters around 100 nm and a zeta potential of -20 mV. Upon NIR irradiation at 0.7 and 1.0 W/cm2, the particles underwent temperature changes (ΔT) of about 15 and 30 ℃, respectively, and produced a large amount of singlet oxygen, demonstrating their excellent PTT/PDT properties. In ovarian cancer cells, treatment with PDA NPs alone did not induce obvious changes in reactive oxygen species (ROS) production or mitochondrial membrane potential (MTP), but when combined with NIR irradiation, these particles caused a significant increase of ROS and a reduction of MTP (P < 0.001), and such changes were more prominent with high power NIR (P < 0.01). PDA NPs alone showed no obvious cytotoxicity, but the combination of PDA with NIR irradiation produced potent killing effect on ovarian cancer cells (P < 0.001), and the effect was much stronger with a high power irradiation (P < 0.001). While PDA alone showed no inhibitory effect on tumor cell metastasis, the combined treatment with PDA and NIR irradiation, especially at a high power, significantly suppressed invasion and migration of ovarian cancer cells (P < 0.001). In the tumor-bearing mouse model, the combined treatment, but not PDA alone, produced a significant inhibitory effect on tumor growth (P < 0.001). PDA NPs combined with NIR has a strong anti-tumor effect, suggesting a potential new therapeutic strategy for ovarian cancer.

Rosmarinic acid exerts anti-inflammatory effect and relieves oxidative stress via Nrf2 activation in carbon tetrachloride-induced liver damage.

Rosmarinic acid (RA) has biological and pharmaceutical properties and shows hepatoprotective potential. However, the hepatoprotective mechanism of RA needs to be further elucidated in vivo and in vitro. This study was aimed to evaluate the protective effect of RA on carbon tetrachloride (CCl4)-induced liver injury and elucidate the hepatoprotective mechanism of RA in vivo and in vitro. In vivo, the mice were orally administrated with RA (10, 20, and 40 mg/kg bw) daily for 28 consecutive days, and 1% CCl4 (5 mL/kg bw, dissolved in peanut oil) was used to induce liver injury. In vitro, the big rat liver (BRL) hepatocytes were pretreated with RA (0.2, 0.4, and 0.8 mg/mL) for 3 h, and then the hepatocytes were treated with CC14 (final concentration, 14 mM) for 3 h to induce cell injury. The related indexes, including hepatic function, oxidative stress, protein expression of nuclear-factor erythroid 2-related factor 2 (Nrf2) pathway, inflammation, histopathological change, hepatocyte apoptosis, and mitochondrial membrane potential, were evaluated. Oral administration of RA to mice considerably decreased the CCl4-induced elevation of serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), triacylglycerols (TG), total cholesterol (TC), total bilirubin (TBIL), hepatic reactive oxygen species (ROS), malondialdehyde (MDA), nitric oxide (NO), 8-hydroxydeoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8). RA also increased the levels of hepatic glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) and the protein expressions of Nrf2, quinine oxidoreductase (NQO1), and heme oxygenease-1 (HO-1). Histopathological examinations indicated that RA (20 and 40 mg/kg bw) alleviated the liver tissue injury induced by CCl4. Moreover, RA inhibited the hepatocyte apoptosis caused by CCl4 based on TUNEL assay. In vitro, RA pretreatment remarkably recovered the cell viability and reduced the CCl4-induced elevation of AST, ALT, lactate dehydrogenase (LDH), ROS, and 8-OHdG. Immunohistochemistry staining demonstrated that pretreatment with RA markedly inhibited the expression of IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and Caspase-3 in CCl4-treated hepatocytes. Additionally, RA pretreatment significantly decreased the elevation of mitochondrial membrane potential in CCl4-treated hepatocytes. RA exerted a protective effect against CCl4-induced liver injury in mice through activating Nrf2 signaling pathway, reducing antioxidant damage, suppressing inflammatory response, and inhibiting hepatocyte apoptosis. RA could attenuate BRL hepatocyte ROS production, DNA oxidative damage, inflammatory response, and apoptosis induced by CCl4 exposure.

Osmoelectric siphon models for signal and water dispersal in wounded plants.

When attacked by herbivores, plants produce electrical signals which can activate the synthesis of the defense mediator jasmonate. These wound-induced membrane potential changes can occur in response to elicitors that are released from damaged plant cells. We list plant-derived elicitors of membrane depolarization. These compounds include the amino acid l-glutamate (Glu), a potential ligand for GLUTAMATE RECEPTOR-LIKE (GLR) proteins that play roles in herbivore-activated electrical signaling. How are membrane depolarization elicitors dispersed in wounded plants? In analogy with widespread turgor-driven cell and organ movements, we propose osmoelectric siphon mechanisms for elicitor transport. These mechanisms are based on membrane depolarization leading to cell water shedding into the apoplast followed by membrane repolarization and water uptake. We discuss two related mechanisms likely to occur in response to small wounds and large wounds that trigger leaf-to-leaf electrical signal propagation. To reduce jasmonate pathway activation, a feeding insect must cut through tissues cleanly. If their mandibles become worn, the herbivore is converted into a robust plant defense activator. Our models may therefore help to explain why numerous plants produce abrasives which can blunt herbivore mouthparts. Finally, if verified, the models we propose may be generalizable for cell to cell transport of water and pathogen-derived regulators.

DHODH: A Promising New Therapeutic Target in Pediatric T-ALL

Acute T-cell lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy in children and young adults. When compared to B-ALL, T-ALL has a higher rate of induction failure as well as relapse, with very poor overall survival in both scenarios. T-ALL has not benefitted from the same immune-mediated therapies (e.g., anti-CD19 CAR-T cells) and poses an ongoing treatment challenge as well as an unmet therapeutic need. Here, we demonstrate that inhibition of dihydroorotate dehydrogenase (DHODH), an enzyme that converts dihydroorotate to orotate as part of the de novo synthesis of uridine, has a robust anti-leukemia effect in both in vitro and in vivo models of T-ALL. DHODH is ubiquitously-expressed, and inhibition of DHODH (DHODHi) leads to rapid depletion of pyrimidine ribo- and deoxyribonucleotides. A cell's ability to tolerate periods of pyrimidine starvation is dependent on a variety of alternative salvage pathways and is not well-understood. Clinical grade DHODH inhibitors are currently under investigation in trials assessing their effect in myeloid malignancies, where they are also known to have a pre-clinical anti-leukemic effect. We sought to understand the mechanism underlying the sensitivity of T-lymphoblasts to DHODHi, and to develop new DHODHi therapeutic combinations for patients with T-ALL. It is known that the intracellular concentration of nucleotides is high in activated T-lymphocytes, underlying the fact that two low-potency DHODHi's are approved for the treatment of autoimmune T-cell diseases (e.g., teriflunomide for multiple sclerosis and leflunomide for rheumatoid arthritis). We hypothesized that T-cells may have a particular addiction to de novo nucleotide synthesis and that this can be exploited as a vulnerability by the small molecule inhibition of DHODH. We demonstrated that T-ALL is highly sensitive to both chemical and genetic DHODHi using a variety of in vitro models (Figure A). Furthermore, we show that T-lymphoblasts respond to DHODHi by undergoing rapid cell cycle arrest and apoptosis. Additionally, we quantified the changes in intracellular nucleotides using PCA extraction and high-performance liquid chromatography (HPLC). The abundance of UTP, CTP, ATP, and GTP were determined across a panel of T-ALL cell lines at baseline and following treatment with a small molecular inhibitor of DHODH. The depletion of intracellular pyrimidines is rapid, falling to ~30% of baseline at 6 hours and confirming that the extracellular salvage is not able to meet the nucleotide demands of these cells even in the presence of complete media. Of note, the concentration of ATP and GTP are unaffected, confirming the specificity of DHODH on de novo pyrimidine synthesis as well as the viability of the cells over the 24-hour treatment. In several in vivo T-ALL models, we demonstrated that DHODH inhibition (specifically using the small molecule inhibitor brequinar) led to rapid, robust disease response (Figure B). These single agent treatment studies (brequinar, 50 mg/kg given every 3-days) were initiated between day 7 - 14 when the leukemias had engrafted and were established with detectable disease in the bone marrow. Three model systems were used: (1) a genetically engineered mouse model (GEMM) of immature T-ALL in which an activated intracellular NOTCH is expressed by retroviral transduction, (2) a transgenic mouse model of mature T-cell leukemia/lymphoma driven by the HTLV1 protein Tax, and (3) a human PDX from a young man with relapsed/refractory T-ALL. DHODH inhibitor therapy was effective in all three models, though clearly and most dramatically prolonged the survival in the GEMM model, driven by dysregulated NOTCH. We are now focused on elucidating the mechanisms by which T-lymphoblasts are so incredibly sensitive to inhibition of DHODH. New data suggests that changes in mitochondrial membrane potential and mitochondrial mass following exposure to DHODHi may offer additional insights into this sensitivity. We are also studying DHODHi in combination with other approved and experimental agents toward the goal of a new therapeutic combination that is safe, well-tolerated, and efficacious for this patient population. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Sulfone Rhodamines for Voltage Imaging.

Fluorescent indicators that respond to changes in biological membrane potentials provide a powerful complement to existing methods for monitoring neuronal activity. Indicators that absorb and emit in the near infrared window are especially attractive, since lower energy wavelengths excite fewer biological molecules and can penetrate more deeply into biological tissues. In this work, we incorporate sulfone rhodamine chromophores into a voltage-sensitive scaffold in order to generate voltage sensitive fluorophores which absorb and emit above 700 nm. These Sulfone Rhodamine Voltage Reporters (SuRhoVRs) partition into cell membranes and display good sensitivity to membrane potential changes. The most sensitive SuRhoVR derivative also displays excellent photostability and can track membrane potential changes in dissociated rat hippocampal neurons.

Antibacterial Mode of Action of β-Amyrin Promotes Apoptosis-Like Death in Escherichia coli by Producing Reactive Oxygen Species.

β-Amyrin is a pentacyclic triterpene widely distributed in leaves and stems worldwide. The ability of β-amyrin to induce the production of reactive oxygen species (ROS) in microorganisms suggests its potential as an antimicrobial agent. Thus, this study aimed to elucidate the antibacterial mode of action of β-amyrin. We treated Escherichia coli cells with β-amyrin and found that it triggered ROS accumulation. Excessive stress caused by ROS, particularly hydroxyl radicals, induces glutathione (GSH) dysfunction. GSH protects cells from oxidative and osmotic stresses; thus, its dysfunction leads to membrane depolarization. The resultant change in membrane potential leads to the release of apoptotic proteins, such as caspases. The activated caspases-like protein promotes the cleavage of DNA into single strands, which is a hallmark of apoptosis-like death in bacteria. Apoptotic cells usually undergo events such as DNA fragmentation and phosphatidylserine exposure, differentiating them from necrotic cells, and the cells treated with β-amyrin in this study were positive for annexin V and negative for propidium iodide, indicating apoptosis-like death. In conclusion, our findings suggest that the antibacterial mode of action of β-amyrin involves the induction of ROS, which resulted in apoptosis-like death in E. coli.

Experimental Oxidative Stress Breaks Down the Barrier Function of the Corneal Endothelium.

Purpose: The fluid pump and barrier functions of the corneal endothelium maintain stromal deturgescence required for corneal transparency. The effect of oxidative stress, a hallmark of Fuchs endothelial corneal dystrophy (FECD), on the endothelial barrier function has been investigated. Methods: The endothelium of porcine corneas ex vivo was exposed to (1) membrane permeable oxidants (H2O2, 100 μM, 1 h; tert-butyl-hydroperoxide, 100 μM, 1 h), or (2) ultraviolet A (UVA) with photosensitizers for 15 min, riboflavin (50 μM) or tryptophan (Trp) (100 μM). The effects on the apical junction complex were analyzed by (1) immunostaining the perijunctional actomyosin ring (PAMR) and ZO-1 and (2) assessment of paracellular flux of fluorescein isothiocyanate (FITC)-avidin across cultured endothelial cells grown on biotinylated-gelatin film. The extent of oxidative stress was quantified by changes in intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) in addition to lipid peroxidation and release of lactate dehydrogenase (LDH). Results: Both methods of oxidative stress led to the disruption of PAMR and ZO-1 concurrent with changes in ROS levels, depolarization of MMP, increased lipid peroxidation, elevated LDH release, and increased permeability of FITC-avidin. The effects of direct oxidants were opposed by SB-203580 [p38 mitogen-activating protein (MAP) kinase inhibitor; 10 μM]. The damage by UVA+photosensitizers was blocked by extracellular catalase (10,000 U/mL). Conclusions: (1) Acute oxidative stress breaks down the barrier function through destruction of PAMR in a p38 MAP kinase-dependent manner. (2) UVA+photosensitizers elicit the breakdown of PAMR via type I reactions, involving H2O2 release. (3) Blocking the oxidative stress prevents loss of barrier function, which could be helpful in the therapeutics of FECD.

Dynamic analysis of neural signal based on Hodgkin–Huxley model

Acupuncture is an important part of traditional Chinese medicine (TCM). Although the efficacy of acupuncture has been widely accepted, the scientific basis behind it is still lack of exploration. Some experimental studies have shown that acupuncture can lead to changes in cell membrane potential, thus affecting the transmission of nerve signals to a certain extent. The change of cell membrane potential mainly depends on a large number of voltage dependent ion channels on the cell membrane. Relevant studies have proved that such voltage dependent ion channels have certain mechanical sensitivity, but the principle is not clear. Based on the theoretical model of cell action potential excitability proposed by Hodgkin–Huxley, taking rat skeletal muscle cell fibers as an example, this paper makes a rough stability analysis of the nonlinear system, explores the conditions of repeated discharge of cell membrane, and simulates the law of cell membrane potential frequency variation under different stimulating currents by numerical calculation. Based on the results of numerical simulation, we propose a hypothesis: Mechanical stimulation will produce a certain amount of stimulating current on the cell membrane, making the cell membrane potential in the state of repeated discharge, so as to block or interfere with the transmission of nerve signals and achieve the analgesic effect and so forth.