Endocytosis of Synaptic Vesicle in Motor Nerve Endings of FUS Transgenic Mice with a Model of Amyotrophic Lateral Sclerosis.

In experiments on the motor nerve endings of the diaphragm of transgenic FUS mice with a model of amyotrophic lateral sclerosis at the pre-symptomatic stage of the disease, the processes of transmitter release and endocytosis of synaptic vesicles were studied. In FUS mice, the intensity of transmitter release during high-frequency stimulation of the motor nerve (50 imp/sec) was lowered. At the same duration of stimulation, the loading of fluorescent dye FM1-43 was lower in FUS mice. However, at the time of stimulation, during which an equal number of quanta are released in wild-type and FUS mice, no differences in the intensity of dye loading were found. Thus, endocytosis is not the key factor in the mechanism of synaptic dysfunction in FUS mice at the pre-symptomatic stage.

Differences in Cladonia rangiferina nitrogen uptake capacity between ammonium and nitrate with uptake kinetics.

Atmospheric nitrogen (N) deposition could affect the structure and function of terrestrial plants. Non-N2-fixing lichens are used to monitor atmospheric N deposition because they rely on the deposited inorganic N (i.e., ammonium and nitrate) as N sources. However, the uptake capacities of lichen on ammonium and nitrate remain unclear, which hinders the application of lichen N content to accurate bioindication of atmospheric N deposition levels. We investigated ammonium and nitrate uptake capacities of Cladonia rangiferina, which was treated with ammonium alone, nitrate alone, and ammonium and nitrate mixture solutions with different mixing ratios under light and dark conditions. The results showed that N uptake rates increased with ammonium and nitrate concentrations in solutions and generally followed the Michaelis-Menten saturation kinetics. Ammonium uptake of C. rangiferina showed higher values of affinity, and was more efficient than the nitrate uptake. Both rates and amounts of nitrate uptake decreased with increasing ratios of ammonium to nitrate in solutions, while ammonium uptake showed no substantial variations, indicating an inhibition of ammonium on nitrate uptake capability. The darkness significantly decreased the maximum uptake rate and efficiency of nitrate, but had much weaker effects on lichen ammonium uptake. These findings highlight the preference of lichen on ammonium as a key N uptake strategy. It is thus necessary to consider the main types of atmospheric inorganic N deposition when using lichens to monitor atmospheric N pollution levels and evaluate N deposition based on lichen ecophysiology.

Comparative effects of ammonium nitrogen on perchlorate degradation performance under heterotrophic condition with different carbon sources.

Perchlorate (ClO4-) mainly exists in the form of ammonium perchlorate in industrial production. However, the degradation mechanisms of different concentrations of ammonium nitrogen (NH4+-N) and ClO4- mixed pollutants in the environment are not well understood. This study aims to explore the potential of different types of carbon sources for ClO4- and NH4+-N biodegradation. Experimental results showed that the concentration and type of carbon sources are decisive to simultaneous removal of NH4+-N and ClO4-. Under condition of C(COD)/C(ClO4-) ratio of 21.15 ± 4.40, the simultaneously removal efficiency of ClO4- and NH4+-N in acetate (Ace) was relatively higher than that in methanol (Met). C(NH4+-N)/C(ClO4-) ratio of 9.66 ± 0.51 and C(COD)/C(ClO4-) ratio of 2.51 ± 0.87 promoted ClO4- reduction in glucose-C (Glu-C). However, high concentration of Glu could cause pH decrease (from 7.57 to 4.59), thereby inhibiting ClO4- reduction. High-throughput sequencing results indicated that Proteobacteria and Bacteroidetes have made a major contribution to the simultaneous removal of NH4+-N and ClO4-. They are two representative bacterial phyla for participating in both ClO4- reduction and denitrification. Notably, the abundance of main ClO4- degrading bacteria (such as Proteobacteria, Chloroflexi, and Firmicutes) significantly increased by 528.57 % in Glu-C. It can be inferred that the concentration of carbon source and NH4+-N were the most important factors determining the removal efficiency of ClO4- by influencing changes in the core microbial community. This study will provide new techniques and mechanistic insights for the simultaneous removal of mixed ClO4- and nitrogen pollutants, which can also provide theoretical support for innovation in future biological treatment processes.

Peripheral positions encode transport specificity in the small multidrug resistance exporters.

In secondary active transporters, a relatively limited set of protein folds have evolved diverse solute transport functions. Because of the conformational changes inherent to transport, altering substrate specificity typically involves remodeling the entire structural landscape, limiting our understanding of how novel substrate specificities evolve. In the current work, we examine a structurally minimalist family of model transport proteins, the small multidrug resistance (SMR) transporters, to understand the molecular basis for the emergence of a novel substrate specificity. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to the activity of a clade of multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this engineered activity. Using X-ray crystallography, solid-supported membrane electrophysiology, binding assays, and a proteoliposome-based quaternary ammonium antiseptic transport assay that we developed, we dissect the mechanistic contributions of these residues to substrate polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate but through mutations peripheral to the binding pocket. Our work provides molecular insight into substrate promiscuity among the SMRs and can be applied to understand multidrug export and the evolution of novel transport functions more generally.

Anaerobic biodegradation enables zero liquid discharge of two-stage nanofiltration system for microelectronic wastewater treatment.

Nanofiltration (NF) is a promising technology in the treatment of microelectronic wastewater. However, the treatment of concentrate derived from NF system remains a substantial technical challenge, impeding the achievement of the zero liquid discharge (ZLD) goal in microelectronic wastewater industries. Herein, a ZLD system, coupling a two-stage NF technology with anaerobic biotechnology was proposed for the treatment of tetramethylammonium hydroxide (TMAH)-contained microelectronic wastewater. The two-stage NF system exhibited favorable efficacy in the removal of conductivity (96 %), total organic carbon (TOC, 90 %), and TMAH (96 %) from microelectronic wastewater. The membrane fouling of this system was dominated by organic fouling, with the second stage NF membrane experiencing a more serious fouling compared to the first stage membrane. The anaerobic biotechnology achieved a near-complete removal of TMAH and an 80 % reduction in TOC for the first stage NF concentrate. Methyloversatilis was the key genus involved in the anaerobic treatment of the microelectronic wastewater concentrate. Specific genes, including dmd-tmd, mtbA, mttB and mttC were identified as significant players in mediating the dehydrogenase and methyl transfer pathways during the process of TMAH biodegradation. This study highlights the potential of anaerobic biodegradation to achieve ZLD in the treatment of TMAH-contained microelectronic wastewater by NF system.

Bioconcentration potential of ionic liquids: New data on membrane partitioning and its comparison with predictions obtained by COSMOmic.

Ionic liquids (ILs) have recently gained significant attention in both the scientific community and industry, but there is a limited understanding of the potential risks they might pose to the environment and human health, including their potential to accumulate in organisms. While membrane and storage lipids have been considered as primary sorption phases driving bioaccumulation, in this study we used an in vitro tool known as solid-supported lipid membranes (SSLMs) to investigate the affinity of ILs to membrane lipid - phosphatidylcholine and compare the results with an existing in silico model. Our findings indicate that ILs may have a strong affinity for the lipids that form cell membranes, with the key factor being the length of the cation's side chain. For quaternary ammonium cations, increase in membrane affinity (logMA) was observed from 3.45 ± 0.06 at 10 carbon atoms in chain to 4.79 ± 0.06 at 14 carbon atoms. We also found that the anion can significantly affect the membrane partitioning of the cation, even though the anions themselves tend to have weaker interactions with phospholipids than the cations of ILs. For 1-methyl-3-octylimidazolium cation the presence of tricyanomethanide anion caused increase in logMA to 4.23 ± 0.06. Although some of our data proved to be consistent with predictions made by the COSMOmic model, there are also significant discrepancies. These results suggest that further research is needed to improve our understanding of the mechanisms and structure-activity relationships involved in ILs bioconcentration and to develop more accurate predictive models.

Bitter Taste Receptor Agonist Denatonium Inhibits Stemness Characteristics in Hematopoietic Stem/Progenitor Cells.

Bone marrow microenvironmental stimuli profoundly impact hematopoietic stem cell fate and biology. As G protein-coupled receptors, the bitter taste receptors (TAS2Rs) are key in transmitting extracellular stimuli into an intracellular response, within the oral cavity but also in extraoral tissues. Their expression in the bone marrow (BM)-derived cells suggests their involvement in sensing the BM microenvironmental fluctuation. In the present study, we demonstrated that umbilical cord blood (UCB)-derived CD34+ cells express fully functional TAS2Rs along with the signal transduction cascade components and their activation by the prototypical agonist, denatonium benzoate, significantly modulated genes involved in stemness maintenance and regulation of cell trafficking. The activation of these specific pathways was confirmed in functional in vitro experiments. Denatonium exposure exerted an antiproliferative effect on UCB-derived CD34+ cells, mainly affecting the most undifferentiated progenitor frequency. It also reduced their clonogenicity and repopulating potential in vitro. In addition, the TAS2R signaling activation impaired the UCB-derived CD34+ cell trafficking, mainly reducing the migration toward the chemoattractant agent CXCL12 and modulating the expression of the adhesion molecules CD62L, CD49d, and CD29. In conclusion, our results in UCB-derived CD34+ cells expand the observation of TAS2R expression in the setting of BM-resident cells and shed light on the role of TAS2Rs in the extrinsic regulation of hematopoietic stem cell functions.

Hypo-osmotic Stress Increases Permeability of Individual Barriers in Escherichia coli Cell Envelope, Enabling Rapid Drug Transport.

Survival of foodborne Gram-negative bacteria during osmotic stress often leads to multidrug resistance development. However, despite the concern, how osmoadaptation alters drug penetration across the Gram-negative bacterial cell envelope has remained inconclusive for years. Here, we have investigated drug permeation and accumulation inside hypo-osmotically shocked Escherichia coli. Three different quaternary ammonium compounds (QACs) are used as cationic amine-containing drug representatives; they also serve as envelope permeability indicators in different assays. Propidium iodide fluorescence reveals cytoplasmic accumulation and overall envelope permeability, while crystal violet sorption and second harmonic generation (SHG) spectroscopy reveal periplasmic accumulation and outer membrane permeability. Malachite green sorption and SHG results reveal transport across both the outer and inner membranes and accumulation in the periplasm as well as cytoplasm. The findings are found to be complementary to one another, collectively revealing enhanced permeabilities of both membranes and the periplasmic space in response to hypo-osmotic stress in E. coli. Enhanced permeability leads to faster QACs transport and higher accumulation in subcellular compartments, whereas transport and accumulation both are negligible under isosmotic conditions. The QACs' transport rates are found to be highly influenced by the osmolytes used, where phosphate ion emerges as a key facilitator of transport across the periplasm into the cytoplasm. E. coli is found viable, with morphology unchanged under extreme hypo-osmotic stress; i.e., it adapts to the situation. The outcome shows that the hypo-osmotic shock to E. coli, specifically using phosphate as an osmolyte, can be beneficial for drug delivery.

Anaerobic hydrolysis of recalcitrant tetramethylammonium from semiconductor wastewater: Performance and mechanisms.

The treatment of tetramethylammonium hydroxide (TMAH)-bearing wastewater, generated in the electronic and semiconductor industries, raises significant concerns due to the neurotoxic, recalcitrant, and bio-inhibiting effects of TMAH. In this study, we proposed the use of an anaerobic hydrolysis bioreactor (AHBR) for TMAH removal, achieving a high removal efficiency of approximately 85%, which greatly surpassed the performance of widely-used advanced oxidation processes (AOPs). Density functional theory calculations indicated that the unexpectedly poor efficiency (5.8-8.0%) of selected AOPs can be attributed to the electrostatic repulsion between oxidants and the tightly bound electrons of TMAH. Metagenomic analyses of the AHBR revealed that Proteobacteria and Euryarchaeota played a dominant role in the transformation of TMAH through processes such as methyl transfer, methanogenesis, and acetyl-coenzyme A synthesis, utilizing methyl-tetrahydromethanopterin as a substrate. Moreover, several potential functional genes (e.g., mprF, basS, bcrB, sugE) related to TMAH resistance have been identified. Molecular docking studies between five selected proteins and tetramethylammonium further provided evidence supporting the roles of these potential functional genes. This study demonstrates the superiority of AHBR as a pretreatment technology compared to several widely-researched AOPs, paving the way for the proper design of treatment processes to abate TMAH in semiconductor wastewater.

Therapeutic potential of otilonium bromide against Vibrio vulnificus.

New drugs are urgently required for the treatment of infections due to an increasing number of new strains of diseases-causing pathogens and antibiotic-resistant bacteria. A library of drugs approved by Food and Drug Administration was screened for efficacy against Vibrio vulnificus using antimicrobial assays. We found that otilonium bromide showed potent antimicrobial activity against V.vulnificus and had a synergistic effect in combination with antibiotics. Field emission transmission electron microscope images revealed that otilonium bromide caused cell division defects in V.vulnificus. Moreover, it significantly inhibited V.vulnificus swarming motility and adhesion to host cells at concentrations lower than the minimum inhibitory concentration. To investigate its inhibitory action mechanisms, we examined the effect of otilonium bromide on the expression levels of several proteins crucial for V.vulnificus growth, motility, and adhesion. It decreased the protein expression levels of cAMP receptor protein and flagellin B, but not HlyU or OmpU. In addition, otilonium bromide significantly decreased the expression levels of outer membrane protein TolCV1, thus inhibiting RtxA1 toxin secretion and substantially reducing V.vulnificus cytotoxicity to host cells. Collectively, these findings suggest that otilonium bromide may be considered as a promising candidate for treating V.vulnificus infections.

GOx-assisted synthesis of pillar[5]arene based supramolecular polymeric nanoparticles for targeted/synergistic chemo-chemodynamic cancer therapy.

BACKGROUND: Cancer is the most serious world's health problems on the global level and various strategies have been developed for cancer therapy. Pillar[5]arene-based supramolecular therapeutic nano-platform (SP/GOx NPs) was constructed successfully via orthogonal dynamic covalent bonds and intermolecular H-bonds with the assistance of glucose oxidase (GOx) and exhibited efficient targeted/synergistic chemo-chemodynamic cancer therapy. METHODS: The morphology of SP/GOx NPs was characterized by DLS, TEM, SEM and EDS mapping. The cancer therapy efficinecy was investigated both in vivo and in vitro. RESULTS: SP/GOx NPs can load drug molecules (Dox) and modify target molecule (FA-Py) on its surface conveniently. When the resultant FA-Py/SP/GOx/Dox NPs enters blood circulation, FA-Py will target it to cancer cells efficiently, where GOx can catalyst the overexpressed glucose to generate H2O2. Subsequently, the generated H2O2 in cancer cells catalyzed by ferrocene unit to form •OH, which can kill cancer cells. Furthermore, the loaded Dox molecules released under acid microenvironment, which can further achieve chemo-therapy. CONCLUSION: All the experiments showed that the excellent antitumor performance of FA-Py/SP/GOx/Dox NPs, which provided an new method for pillar[5]arene-based supramolecular polymer for biomedical applications.

Ferric Ammonium Citrate Upregulates PD-L1 Expression through Generation of Reactive Oxygen Species.

Iron plays an important role in macrophage polarization by altering metabolic and redox status. However, the impact of iron on the immune status of macrophages is still controversial. In this study, we report that ferric ammonium citrate (FAC) upregulates PD-L1 expression in macrophages. FAC not only altered the phenotype of macrophages but also led to enriching immune-modulatory T cell subsets. Since iron is known to be a constituent of coenzymes facilitating metabolic processes in mitochondria, we examined the metabolic status of FAC-overloaded macrophages by measuring the oxygen consumption rate (OCR) and the represented coenzyme, aconitase. In addition to enhancement of metabolic processes, FAC accelerated the Fenton reaction in macrophages, which also contributed to the facilitation of oxygen consumption. We reasoned that the enhancement of the OCR leads to the production of reactive oxygen species (ROS), which are directly linked to PD-L1 induction. Using ferrostatin, rotenone, and N-acetyl-L-cysteine, we confirmed that metabolic and redox regulation is responsible for FAC-mediated PD-L1 expression. Furthermore, we suggested that FAC-induced ROS production may explain FAC-mediated pro- and anti-inflammatory responses in macrophages. These findings may extend our understanding of regulating iron concentration during immune checkpoint therapy in cancer patients.

Bitter Flavored, Soft Composites for Wearables Designed to Reduce Risks of Choking in Infants.

Wireless, skin-integrated devices for continuous, clinical-quality monitoring of vital signs have the potential to greatly improve the care of patients in neonatal and pediatric intensive-care units. These same technologies can also be used in the home, across a broad spectrum of ages, from beginning to end of life. Although miniaturized forms of such devices minimize patient burden and improve compliance, they represent life-threatening choking hazards for infants. A materials strategy is presented here to address this concern. Specifically, composite materials are introduced as soft encapsulating layers and gentle adhesives that release chemical compounds designed to elicit an intense bitter taste when placed in the mouth. Reflexive reactions to this sensation strongly reduce the potential for ingestion, as a safety feature. The materials systems described involve a non-toxic bitterant (denatonium benzoate) as a dopant in an elastomeric (poly(dimethylsiloxane)) or hydrogel matrix. Experimental and computational studies of these composite materials and the kinetics of release of the bitterant define the key properties. Incorporation into various wireless skin-integrated sensors demonstrates their utility in functional systems. This simple strategy offers valuable protective capabilities, with broad practical relevance to the welfare of children monitored with wearable devices.

Molecular Dynamic Simulation Search for Possible Amphiphilic Drug Discovery for Covid-19.

To determine whether quaternary ammonium (k21) binds to Severe Acute Respiratory Syndrome-Coronavirus 2 (SARS-CoV-2) spike protein via computational molecular docking simulations, the crystal structure of the SARS-CoV-2 spike receptor-binding domain complexed with ACE-2 (PDB ID: 6LZG) was downloaded from RCSB PD and prepared using Schrodinger 2019-4. The entry of SARS-CoV-2 inside humans is through lung tissues with a pH of 7.38-7.42. A two-dimensional structure of k-21 was drawn using the 2D-sketcher of Maestro 12.2 and trimmed of C18 alkyl chains from all four arms with the assumption that the core moiety k-21 was without C18. The immunogenic potential of k21/QA was conducted using the C-ImmSim server for a position-specific scoring matrix analyzing the human host immune system response. Therapeutic probability was shown using prediction models with negative and positive control drugs. Negative scores show that the binding of a quaternary ammonium compound with the spike protein's binding site is favorable. The drug molecule has a large Root Mean Square Deviation fluctuation due to the less complex geometry of the drug molecule, which is suggestive of a profound impact on the regular geometry of a viral protein. There is high concentration of Immunoglobulin M/Immunoglobulin G, which is concomitant of virus reduction. The proposed drug formulation based on quaternary ammonium to characterize affinity to the SARS-CoV-2 spike protein using simulation and computational immunological methods has shown promising findings.

Chronic metformin treatment decreases cardiac injury during ischemia-reperfusion by attenuating endoplasmic reticulum stress with improved mitochondrial function.

Aging impairs mitochondrial function that leads to greater cardiac injury during ischemia and reperfusion. Cardiac endoplasm reticulum (ER) stress increases with age and contributes to mitochondrial dysfunction. Metformin is an anti-diabetic drug that protects cardiac mitochondria during acute ER stress. We hypothesized that metformin treatment would improve preexisting mitochondrial dysfunction in aged hearts by attenuating ER stress, followed by a decrease in cardiac injury during subsequent ischemia and reperfusion. Male young (3 mo.) and aged mice (24 mo.) received metformin (300 mg/kg/day) dissolved in drinking water with sucrose (0.2 g/100 ml) as sweetener for two weeks versus sucrose vehicle alone. Cytosol, subsarcolemmal (SSM), and interfibrillar mitochondria (IFM) were isolated. In separate groups, cardioprotection was evaluated using ex vivo isolated heart perfusion with 25 min. global ischemia and 60 min. reperfusion. Infarct size was measured. The contents of CHOP and cleaved ATF6 were decreased in metformin-treated 24 mo. mice compared to vehicle, supporting a decrease in ER stress. Metformin treatment improved OXPHOS in IFM in 24 mo. using a complex I substrate. Metformin treatment decreased infarct size following ischemia-reperfusion. Thus, metformin feeding decreased cardiac injury in aged mice during ischemia-reperfusion by improving pre-ischemic mitochondrial function via inhibition of ER stress.

Phase Change Dimethyldioctadecylammonium-Shelled Microdroplets as a Promising Drug Delivery System: Results on 3D Spheroids of Mammalian Tumor Cells.

Significant improvement of phase-change perfluorocarbon microdroplets (MDs) in the vast theranostic scenario passes through the optimization of the MDs composition with respect to synthesis efficiency, stability, and drug delivery capability. To this aim, decafluoropentane (DFP) MDs stabilized by a shell of dimethyldioctadecylammonium bromide (DDAB) cationic surfactant were designed. A high concentration of DDAB-MDs was readily obtained within a few seconds by pulsed high-power insonation, resulting in low polydisperse 1 µm size droplets. Highly positive ζ-potential, together with a long, saturated hydrocarbon chains of the DDAB shell, are key factors to stabilize the droplet and the drug cargo therein. The high affinity of the DDAB shell with cell plasma membrane allows for localized chemotherapeutics delivery by increasing the drug concentration at the tumor cell interface and boosting the uptake. This would turn DDAB-MDs into a relevant drug delivery tool exhibiting high antitumor activity at very low drug doses. In this work, the efficacy of such an approach is shown to dramatically improve the effect of doxorubicin against 3D spheroids of mammalian tumor cells, MDA-MB-231. The use of three-dimensional (3D) cell cultures developed in the form of multicellular tumor spheroids (i.e., densely packed cells in a spherical shape) has numerous advantages compared to 2D cell cultures: in addition to have the potential to bridge the gap between conventional in vitro studies and animal testing, it will improve the ability to perform more predictive in vitro screening assays for preclinical drug development or evaluate the potential of off-label drugs and new co-targeting strategies.

Microbial properties of the granular sludge in a psychrophilic UASB reactor fed with electronics industry wastewater containing organic chemicals.

In this study, a lab-scale upflow anaerobic sludge blanket (UASB) reactor was applied to the treatment of artificial electronics industry wastewater containing tetramethylammonium-hydroxide (TMAH), monoethanolamine (MEA), and isopropyl-alcohol (IPA) in order to evaluate process performance and degradation properties. During 800 days of operation, 96% efficiency of chemical oxygen demand (COD) removal was stably achieved at an organic loading rate of 8.5 kgCOD/m3/day at 18-19 °C. MEA degradation, carried out by acid-forming eubacteria, was confirmed within a week. The physical properties of the retained granular sludge were degraded by feeding with TMAH wastewater, but maintained by feeding with MEA wastewater due to an accumulation of species from the genus Methanosaeta and family Geobacteraceae. Analysis of the microbial community structure via SEM and 16S rRNA genes showed a proliferation of Methanomethylovorans-like cells and Methanosaeta-like cells at the surface and in the core of the granular sludge with TMAH, MEA and IPA acclimation. Furthermore, a batch degradation experiment confirmed that process inhibition due to increasing chemical concentration was relatively stronger for TMAH than for MEA or IPA. Thus, controlling the TMAH concentration of the influent to below 1 gCOD/L will be important for the stable treatment of electronics industry wastewater by UASB technology.

Increasing Intracellular Levels of Iron with Ferric Ammonium Citrate Leads to Reduced P-glycoprotein Expression in Human Immortalised Brain Microvascular Endothelial Cells.

PURPOSE: P-glycoprotein (P-gp) at the blood-brain barrier (BBB) precludes the brain penetration of many xenobiotics and mediates brain-to-blood clearance of ß-amyloid, which accumulates in the Alzheimer's disease (AD) brain. Zinc and copper are reported to modulate BBB expression and function of P-gp; however, the impact of exogenous iron, which accumulates in AD, on P-gp dynamics remains unknown. METHODS: P-gp protein and MDR1 transcript levels were assessed in immortalised human cerebral microvascular endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC; 250 µM, 72 h), by Western blotting and RT-qPCR, respectively. P-gp function was assessed using rhodamine-123 and [3H]-digoxin accumulation. Intracellular reactive oxygen species (ROS) levels were determined using 2',7'-dichlorofluorescin diacetate and intracellular iron levels quantified using a ferrozine assay. RESULTS: FAC treatment significantly reduced P-gp protein (36%) and MDR1 mRNA (16%) levels, with no significant change in rhodamine-123 or [3H]-digoxin accumulation. While P-gp/MDR1 downregulation was associated with elevated ROS and intracellular iron, MDR1 downregulation was not attenuated with the antioxidant N-acetylcysteine nor the iron chelators desferrioxamine and deferiprone, suggesting the involvement of a ROS-independent mechanism or incomplete iron chelation. CONCLUSIONS: These studies demonstrate that iron negatively regulates P-gp expression at the BBB, potentially impacting CNS drug delivery and brain ß-amyloid clearance.

Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA.

Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA+) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves as a model to its homologous eukaryotic counterparts. By the combined use of a thermal denaturation assay and the analysis of homo-Förster resonance energy transfer in a mutant channel containing a single tryptophan (W67) per subunit, we found that TOA+ binds the channel cavity with high affinity, either with the inner gate open or closed. Moreover, TOA+ bound at the cavity allosterically shifts the equilibrium of the channel's selectivity filter conformation from conductive to an inactivated-like form. The inactivated TOA+-KcsA complex exhibits a loss in the affinity towards permeant K+ at pH 7.0, when the channel is in its closed state, but maintains the two sets of K+ binding sites and the W67-W67 intersubunit distances characteristic of the selectivity filter in the channel resting state. Thus, the TOA+-bound state differs clearly from the collapsed channel state described by X-ray crystallography and claimed to represent the inactivated form of KcsA.

Efficient clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish.

The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders.