Aconitase Activity Research Articles

Inhibition of prostate cancer proliferation by Deferiprone.

Cancer growth and proliferation rely on intracellular iron availability. We studied the effects of Deferiprone (DFP), a chelator of intracellular iron, on three prostate cancer cell lines: murine, metastatic TRAMP-C2; murine, non-metastatic Myc-CaP; and human, non-metastatic 22rv1. The effects of DFP were evaluated at different cellular levels: cell culture proliferation and migration; metabolism of live cells (time-course multi-nuclear magnetic resonance spectroscopy cell perfusion studies, with 1-13 C-glucose, and extracellular flux analysis); and expression (Western blot) and activity of mitochondrial aconitase, an iron-dependent enzyme. The 50% and 90% inhibitory concentrations (IC50 and IC90 , respectively) of DFP for the three cell lines after 48h of incubation were within the ranges 51-67μM and 81-186μM, respectively. Exposure to 100μM DFP led to: (i) significant inhibition of cell migration after different exposure times, ranging from 12h (TRAMP-C2) to 48h (22rv1), in agreement with the respective cell doubling times; (ii) significantly decreased glucose consumption and glucose-driven tricarboxylic acid cycle activity in metastatic TRAMP-C2 cells, during the first 10h of exposure, and impaired cellular bioenergetics and membrane phospholipid turnover after 23h of exposure, consistent with a cytostatic effect of DFP. At this time point, all cell lines studied showed: (iii) significant decreases in mitochondrial functional parameters associated with the oxygen consumption rate, and (iv) significantly lower mitochondrial aconitase expression and activity. Our results indicate the potential of DFP to inhibit prostate cancer proliferation at clinically relevant doses and plasma concentrations.

Differences in intracellular mobile zinc levels affect susceptibility to plasma-activated medium-induced cytotoxicity

There is growing evidence that plasma-activated medium (PAM), which is prepared by non-thermal plasma (NTP) irradiation of cell-free medium, is a beneficial tool for cancer therapy. PAM has been reported to preferentially kill cancer cells; however, its mechanism is not fully understood. Since PAM contains reactive oxygen species (ROS) and reactive nitrogen species, the anti-cancer effects of PAM are thought to be attributed to oxidative stress induced by these reactive molecules. Oxidative stress has been shown to release zinc (Zn2+) from intracellular Zn2+ stores and provoke Zn2+-dependent cell death. We have previously demonstrated that intracellular free Zn2+ plays a critical role in PAM-induced cell death in human neuroblastoma SH-SY5Y cells. In this study, we found that normal human fibroblasts were less susceptible to PAM cytotoxicity compared with SH-SY5Y cells. PAM decreased intracellular NAD+ levels in both cells, whereas the depletion of ATP and mitochondrial ROS generation was hardly observed in fibroblasts. Intracellular mobile Zn2+ contents of fibroblasts were lower than those of SH-SY5Y cells. PAM suppressed the activity of aconitase, which is a tricarboxylic acid cycle enzyme, only in SH-SY5Y cells, and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a Zn2+ chelator, counteracted the suppression. The combination treatment with PAM and Zn2+ augmented PAM-induced ATP depletion, mitochondrial ROS generation, and cytotoxicity in fibroblasts. These findings suggest the possibility that cells with high intracellular mobile Zn2+ are susceptible to PAM cytotoxicity. Therefore, we concluded that the differences in mobile Zn2+ levels affect PAM-induced cellular responses.

Swimming exercise reverses aging-related contractile abnormalities of female heart by improving structural alterations.

The objective of this study was to examine the effect of swimming exercise on aging-related Ca2+ handling alterations and structural abnormalities of female rat heart. For this purpose, 4-month and 24-month old female rats were used and divided into three following groups: sedentary young (SY), sedentary old (SO), and exercised old (Ex-O). Swimming exercise was performed for 8 weeks (60 min/day, 5 days/week). Myocyte shortening, L-type Ca2+ currents and associated Ca2+ transients were measured from ventricular myocytes at 36 ± 1°C. NOX-4 levels, aconitase activity, glutathione measurements and ultrastructural examination by electron microscopy were conducted in heart tissue. Swimming exercise reversed the reduced shortening and slowed kinetics of aged cardiomyocytes. Although the current density was similar for all groups, Ca2+ transients were higher in SO and Ex-O myocytes with respect to the SY group. Caffeine-induced Ca2+ transients and the integrated NCX current were lower in cardiomyocytes of SY rats compared with other groups, suggesting an increased sarcoplasmic reticulum Ca2+ content in an aged heart. Aging led to upregulated cardiac NOX-4 along with declined aconitase activity. Although it did not reverse these oxidative parameters, swimming exercise achieved a significant increase in glutathione levels and improved structural alterations of old rats' hearts. We conclude that swimming exercise upregulates antioxidant defense capacity and improves structural abnormalities of senescent female rat heart, although it does not change Ca2+ handling alterations further. Thereby, it improves contractile function of aged myocardium by mitigating detrimental effects of oxidative stress.

Distinct Effects of miR-210 Reduction on Neurogenesis: Increased Neuronal Survival of Inflammation But Reduced Proliferation Associated with Mitochondrial Enhancement.

Neurogenesis is essential to brain development and plays a central role in the response to brain injury. Stroke and head trauma stimulate proliferation of endogenous neural stem cells (NSCs); however, the survival of young neurons is sharply reduced by postinjury inflammation. Cellular mitochondria are critical to successful neurogenesis and are a major target of inflammatory injury. Mitochondrial protection was shown to improve survival of young neurons. This study tested whether reducing cellular microRNA-210 (miR-210) would enhance mitochondrial function and improve survival of young murine neurons under inflammatory conditions. Several studies have demonstrated the potential of miR-210 inhibition to enhance and protect mitochondrial function through upregulation of mitochondrial proteins. Here, miR-210 inhibition significantly increased neuronal survival and protected the activity of mitochondrial enzymes cytochrome c oxidase and aconitase in differentiating NSC cultures exposed to inflammatory mediators. Unexpectedly, we found that reducing miR-210 significantly attenuated NSC proliferation upon induction of differentiation. Further investigation revealed that increased mitochondrial function suppressed the shift to primarily glycolytic metabolism and reduced mitochondrial length characteristic of dividing cells. Activation of AMP-regulated protein kinase-retinoblastoma signaling is important in NSC proliferation and the reduction of this activation observed by miR-210 inhibition is one mechanism contributing to the reduced proliferation. Postinjury neurogenesis occurs as a burst of proliferation that peaks in days, followed by migration and differentiation over weeks. Our studies suggest that mitochondrial protective miR-210 inhibition should be delayed until after the initial burst of proliferation, but could be beneficial during the prolonged differentiation stage.SIGNIFICANCE STATEMENT Increasing the success of endogenous neurogenesis after brain injury holds therapeutic promise. Postinjury inflammation markedly reduces newborn neuron survival. This study found that enhancement of mitochondrial function by reducing microRNA-210 (miR-210) levels could improve survival of young neurons under inflammatory conditions. miR-210 inhibition protected the activity of mitochondrial enzymes cytochrome c oxidase and aconitase. Conversely, we observed decreased precursor cell proliferation likely due to suppression of the AMP-regulated protein kinase-retinoblastoma axis with miR-210 inhibition. Therefore, mitochondrial protection is a double-edged sword: early inhibition reduces proliferation, but inhibition later significantly increases neuroblast survival. This explains in part the contradictory published reports of the effects of miR-210 on neurogenesis.

The Active Component of Aspirin, Salicylic Acid, Promotes Staphylococcus aureus Biofilm Formation in a PIA-dependent Manner.

Aspirin has provided clear benefits to human health. But salicylic acid (SAL) -the main aspirin biometabolite- exerts several effects on eukaryote and prokaryote cells. SAL can affect, for instance, the expression of Staphylococcus aureus virulence factors. SAL can also form complexes with iron cations and it has been shown that different iron chelating molecules diminished the formation of S. aureus biofilm. The aim of this study was to elucidate whether the iron content limitation caused by SAL can modify the S. aureus metabolism and/or metabolic regulators thus changing the expression of the main polysaccharides involved in biofilm formation. The exposure of biofilm to 2 mM SAL induced a 27% reduction in the intracellular free Fe2+ concentration compared with the controls. In addition, SAL depleted 23% of the available free Fe2+ cation in culture media. These moderate iron-limited conditions promoted an intensification of biofilms formed by strain Newman and by S. aureus clinical isolates related to the USA300 and USA100 clones. The slight decrease in iron bioavailability generated by SAL was enough to induce the increase of PIA expression in biofilms formed by methicillin-resistant as well as methicillin-sensitive S. aureus strains. S. aureus did not produce capsular polysaccharide (CP) when it was forming biofilms under any of the experimental conditions tested. Furthermore, SAL diminished aconitase activity and stimulated the lactic fermentation pathway in bacteria forming biofilms. The polysaccharide composition of S. aureus biofilms was examined and FTIR spectroscopic analysis revealed a clear impact of SAL in a codY-dependent manner. Moreover, SAL negatively affected codY transcription in mature biofilms thus relieving the CodY repression of the ica operon. Treatment of mice with SAL induced a significant increase of S aureus colonization. It is suggested that the elevated PIA expression induced by SAL might be responsible for the high nasal colonization observed in mice. SAL-induced biofilms may contribute to S. aureus infection persistence in vegetarian individuals as well as in patients that frequently consume aspirin.

TLR4 knockout attenuated high fat diet-induced cardiac dysfunction via NF-κB/JNK-dependent activation of autophagy

Obesity is commonly associated with a low grade systemic inflammation, which may contribute to the onset and development of myocardial remodeling and contractile dysfunction. Toll-like receptor 4 (TLR4) plays an important role in innate immunity and inflammation although its role in high fat diet-induced obesity cardiac dysfunction remains elusive. This study was designed to examine the effect of TLR4 ablation on high fat diet intake-induced cardiac anomalies, if any, and underlying mechanism(s) involved. Wild-type (WT) and TLR4 knockout mice were fed normal or high fat (60% calorie from fat) diet for 12weeks prior to assessment of mechanical and intracellular Ca2+ properties. The inflammatory signaling proteins (TLR4, NF-κB, and JNK) and autophagic markers (Atg5, Atg12, LC3B and p62) were evaluated. Our results revealed that high fat diet intake promoted obesity, marked decrease in fractional shortening, and cardiomyocyte contractile capacity with dampened intracellular Ca2+ release and clearance, elevated ROS generation and oxidative stress as measured by aconitase activity, the effects of which were significantly attenuated by TLR4 knockout. In addition, high fat intake downregulated levels of Atg5, Atg12 and LC3B, while increasing p62 accumulation. TLR4 knockout itself did not affect Atg5, Atg12, LC3B and p62 levels while it reconciled high fat diet intake-induced changes in autophagy. In addition, TLR4 knockout alleviated high fat diet-induced phosphorylation of IKKβ, JNK and mTOR. In vitro study revealed that palmitic acid suppressed cardiomyocyte contractile function, the effect of which was inhibited the TLR4 inhibitor CLI-095, the JNK inhibitor AS601245 or the NF-κB inhibitor Celastrol. Taken together, these data showed that TLR4 knockout ameliorated high fat diet-induced cardiac contractile and intracellular Ca2+ anomalies through inhibition of inflammation and ROS, possibly through a NF-κB/JNK-dependent activation of autophagy. This article is part of a Special Issue entitled: Genetic and epigenetic control of heart failure - edited by Jun Ren & Megan Yingmei Zhang.

Oxidative Stress and Amyloid Toxicity: Insights From Yeast.

Alzheimer's disease is the most common neurodegenerative disorder. One of the factors that promotes neurodegeneration is the accumulation of senile plaques formed by Aβ peptide. In this paper, it was analyzed that if oxidative stress is cause or consequence of amyloid cascade and the role of antioxidant defense system in this process, using S. cerevisiae (with a multicopy plasmid containing the Aβ1-42 sequence) as experimental model. Cells grown on glycerol were more tolerant than when grown on glucose, strengthening the role of the antioxidant defense system against Aβ accumulation. Antioxidant defense deficiency did not change the pattern of amyloid aggregation. On the other hand, the presence of Aβ increased the level of intracellular oxidation and induced the activity of catalase, superoxide dismutase, and aconitase. Peroxissomal catalase deficient cells (Δcta1), were more sensitive to Aβ toxicity than the wild type strain, while mitochondrial superoxide dismutase (Sod2) deficient cells displayed the highest frequency of petites. Besides, Aβ alters the oxygen consumption and the activity of complex III and IV. Taken together, our results point out that the Aβ toxicity mechanism involves an oxidative stress induction by increasing ROS production into the mitochondria, where Cta1 and Sod2 play a crucial role in the regulation of the redox balance. J. Cell. Biochem. 118: 1442-1452, 2017. © 2016 Wiley Periodicals, Inc.

Production of itaconate by whole-cell bioconversion of citrate mediated by expression of multiple cis-aconitate decarboxylase (cadA) genes in Escherichia coli

Itaconate, a C5 unsaturated dicarboxylic acid, is an important chemical building block that is used in manufacturing high-value products, such as latex and superabsorbent polymers. Itaconate is produced by fermentation of sugars by the filamentous fungus Aspergillus terreus. However, fermentation by A. terreus involves a long fermentation period and the formation of various byproducts, resulting in high production costs. E. coli has been developed as an alternative for producing itaconate. However, fermentation of glucose gives low conversion yields and low productivity. Here, we report the whole-cell bioconversion of citrate to itaconate with enhanced aconitase and cis-aconitate decarboxylase activities by controlling the expression of multiple cadA genes. In addition, this bioconversion system does not require the use of buffers, which reduces the production cost and the byproducts released during purification. Using this whole-cell bioconversion system, we were able to catalyze the conversion of 319.8 mM of itaconate (41.6 g/L) from 500 mM citrate without any buffer system or additional cofactors, with 64.0% conversion in 19 h and a productivity of 2.19 g/L/h. Our bioconversion system suggests very high productivity for itaconate production.

Ontogenetic Approach to the Study of Mechanisms of Copper-Induced Liver Fibrosis

On the model of Cu-induced liver fibrosis, the relationship between the activity of prooxidant-antioxidant system, immune system parameters, liver morphology and several physiological parameters (body temperature and performance ability of the animals, taking into account their ages) was investigated. Classical biochemical, immunological, histological and physiological methods of investigation were used. The subjects of the study were male Wistar rats of 3-month (young) and 20-month (old) age. For the induction of liver fibrosis, experimental animals were successively injected with copper sulfate (three times at intervals of 24 hours at a dose corresponding to 33% of the lethal one). It was shown that after five days of sulfate copper administration inflammatory reactions in the liver, damage of the vessel epithelium, an increase in collagen content, and other morphological changes were detected. At this time, the content of lipid hydroperoxides in the liver and blood serum was increased, the activity of a number of antioxidant enzymes was reduced, and the activity of aconitase was two times less compared to values in the control group. These changes correlated with a decrease in the amount and activity of phagocytic cells in the blood of experimental animals. Inhibition of the general metabolism was accompanied by a decrease in body temperature, loss of body weight and performance ability. The relationship between a specific metabolic pattern in animals with Cu-induced fibrosis was age-dependent. The formed specific adaptive metabolic pattern is unstable, and in the future it can be realized in one of three possible adaptive strategies, the choice of which is influenced by age.

Tumor-initiating cells of breast and prostate origin show alterations in the expression of genes related to iron metabolism

The importance of iron in the growth and progression of tumors has been widely documented. In this report, we show that tumor-initiating cells (TICs), represented by spheres derived from the MCF7 cell line, exhibit higher intracellular labile iron pool, mitochondrial iron accumulation and are more susceptible to iron chelation. TICs also show activation of the IRP/IRE system, leading to higher iron uptake and decrease in iron storage, suggesting that level of properly assembled cytosolic iron-sulfur clusters (FeS) is reduced. This finding is confirmed by lower enzymatic activity of aconitase and FeS cluster biogenesis enzymes, as well as lower levels of reduced glutathione, implying reduced FeS clusters synthesis/utilization in TICs. Importantly, we have identified specific gene signature related to iron metabolism consisting of genes regulating iron uptake, mitochondrial FeS cluster biogenesis and hypoxic response (ABCB10, ACO1, CYBRD1, EPAS1, GLRX5, HEPH, HFE, IREB2, QSOX1 and TFRC). Principal component analysis based on this signature is able to distinguish TICs from cancer cells in vitro and also Leukemia-initiating cells (LICs) from non-LICs in the mouse model of acute promyelocytic leukemia (APL). Majority of the described changes were also recapitulated in an alternative model represented by MCF7 cells resistant to tamoxifen (TAMR) that exhibit features of TICs. Our findings point to the critical importance of redox balance and iron metabolism-related genes and proteins in the context of cancer and TICs that could be potentially used for cancer diagnostics or therapy.

Polyphenol-rich virgin olive oil reduces insulin resistance and liver inflammation and improves mitochondrial dysfunction in high-fat diet fed rats.

Virgin olive oil is an essential component of the Mediterranean diet. Its antioxidant and anti-inflammatory properties are mainly linked to phenolic contents. This study aims to evaluate the beneficial effects of a polyphenol-rich virgin olive oil (HPCOO) or olive oil without polyphenols (WPOO) in rats fed high-fat diet (HFD). Male Sprague-Dawley rats were divided into four groups based on the different types of diet: (I) standard diet (STD); (II) HFD; (III) HFD containing WPOO, and (IV) HFD containing HPCOO. HPCOO and WPOO induced a significant improvement of HFD-induced impaired glucose homeostasis (by hyperglycemia, altered oral glucose tolerance, and HOMA-IR) and inflammatory status modulating pro- and anti-inflammatory cytokines (TNF-α, IL-1, and IL-10) and adipokines. Moreover, HPCOO and less extensively WPOO, limited HFD-induced liver oxidative and nitrosative stress and increased hepatic fatty acid oxidation. To study mitochondrial performance, oxidative capacity and energy efficiency were also evaluated in isolated liver mitochondria. HPCOO, but not WPOO, reduced H2 O2 release and aconitase activity by decreasing degree of coupling, which plays a major role in the control of mitochondrial reactive oxygen species emission. HPCOO limits HFD-induced insulin resistance, inflammation, and hepatic oxidative stress, preventing nonalcoholic fatty liver disease progression.

Inhibitory effect of homocysteine on rat neural stem cell growth in vitro is associated with reduced protein levels and enzymatic activities of aconitase and respiratory complex III.

Increased blood plasma concentration of the sulphur amino acid homocysteine (Hcy) is considered as an independent risk factor of the neurodegenerative diseases. However, the detailed molecular mechanisms by which Hcy leads to neurotoxicity have yet to be clarified. Recent research has suggested that neurotoxicity of Hcy may involve negative regulation of neural stem cell (NSC) proliferation. In the current study, primary NSCs were isolated from neonatal rat brain hippocampus and the inhibition in cell growth was observed after exposure to l50μM and 500μML-Hcy. The changes in protein expression were monitored with densitometric 2D-gel electrophoresis coupled with MALDI-TOF mass spectrometry. Proteomic analysis revealed that the expression levels of two mitochondrial proteins, cytochrome bc1 complex2 (UQCRC2, the major component of electron transport chain complex III) and aconitase (an enzyme involved in the tricarboxylic acid cycle), were decreased in Hcy treatment group, compared to control group. Protein expression was further verified by Western blot, and their enzymatic activities were also down-regulated in NSCs after Hcy treatment. Restoration of aconitase and UQCRC2 protein levels using their expression vectors could partly rescue the cell viability inhibition caused by Hcy. Moreover, Hcy caused the increase in the intracellular levels of reactive oxygen species (ROS) and the decrease in ATP content, which are known to play important roles in the cellular stress response of the cell growth. Altogether, the results suggest that the decreased expression and enzymatic activities of the mitochondrial proteins may be possible causes of the overproduction of ROS and depletion of ATP. The inhibition in cell growth at the end of Hcy treatment was probably due to the changes in protein expression and mitochondrial dysfunction in vitro cultures of NSCs.

Regulation of the Erythroid Iron Deprivation Response By a TfR2-Scribble-Epor Integrated Module Coupling Iron Sensing with Receptor Presentation

Iron deficiency in humans causes desensitization of erythroid progenitors to erythropoietin, the principal cytokine for erythroid survival, proliferation, and differentiation. This nutrient deprivation response acts in a lineage-selective, non-apoptotic manner to restrain expansion of the tissue responsible for the majority of iron consumption, thereby triaging iron utilization under conditions of deficiency. The molecular basis for this response is incompletely understood but involves transferrin receptor 2 (TfR2) and aconitase enzymatic activity as extra- and intracellular iron sensors. Here, we identify a multi-component, integrated module connecting these two elements of erythroid iron sensing with endo-lysosomal trafficking, receptor surface delivery, and configuration of EpoR signaling. Specifically, iron and aconitase activity modulate the rate of TfR2 catabolism by cathepsin protease activity. Scribble, a conserved regulator of receptor transport and signaling, binds TfR2 and serves as a downstream effector through to its co-catabolism. Scribble levels in turn influence surface delivery of the erythropoietin receptor (EpoR), which physically interacts with both Scribble and TfR2. The TfR2-dependent downregulation of Scribble associated with iron deprivation diminishes surface EpoR density and skews its signaling from JAK-STAT to Akt pathways. Mice with surface-trapped, endocytosis-defective EpoR fail to develop anemia in response to iron deficiency. These findings establish a tissue-specific nutrient sensing pathway relevant to the pathogenesis of human anemias and their resistance to erythropoietin therapy. DisclosuresNo relevant conflicts of interest to declare.

Real-Time Fluorescence Measurements of ROS and [Ca2+] in Ischemic / Reperfused Rat Hearts: Detectable Increases Occur only after Mitochondrial Pore Opening and Are Attenuated by Ischemic Preconditioning.

Mitochondrial permeability transition pore (mPTP) opening is critical for ischemia / reperfusion (I/R) injury and is associated with increased [Ca2+] and reactive oxygen species (ROS). Here we employ surface fluorescence to establish the temporal sequence of these events in beating perfused hearts subject to global I/R. A bespoke fluorimeter was used to synchronously monitor surface fluorescence and reflectance of Langendorff-perfused rat hearts at multiple wavelengths, with simultaneous measurements of hemodynamic function. Potential interference by motion artefacts and internal filtering was assessed and minimised. Re-oxidation of NAD(P)H and flavoproteins on reperfusion (detected using autofluorescence) was rapid (t0.5 < 15 s) and significantly slower following ischemic preconditioning (IP). This argues against superoxide production from reduced Complex 1 being a critical mediator of initial mPTP opening during early reperfusion. Furthermore, MitoPY1 (a mitochondria-targeted H2O2-sensitive fluorescent probe) and aconitase activity measurements failed to detect matrix ROS increases during early reperfusion. However, two different fluorescent cytosolic ROS probes did detect ROS increases after 2–3 min of reperfusion, which was shown to be after initiation of mPTP opening. Cyclosporin A (CsA) and IP attenuated these responses and reduced infarct size. [Ca2+]i (monitored with Indo-1) increased progressively during ischemia, but dropped rapidly within 90 s of reperfusion when total mitochondrial [Ca2+] was shown to be increased. These early changes in [Ca2+] were not attenuated by IP, but substantial [Ca2+] increases were observed after 2–3 min reperfusion and these were prevented by both IP and CsA. Our data suggest that the major increases in ROS and [Ca2+] detected later in reperfusion are secondary to mPTP opening. If earlier IP-sensitive changes occur that might trigger initial mPTP opening they are below our limit of detection. Rather, we suggest that IP may inhibit initial mPTP opening by alternative mechanisms such as prevention of hexokinase 2 dissociation from mitochondria during ischemia.

A novel mutation in nuclear prelamin a recognition factor-like causes diffuse pulmonary arteriovenous malformations.

Two daughters in a Chinese consanguineous family were diagnosed as diffuse pulmonary arteriovenous malformations (PAVMs) and screened using whole exome sequencing (WES) and copy number variations (CNVs) chips. Though no mutation was found in the established causative genes of capillary malformation-AVMs (CM-AVMs) or PAVMs, Ser161Ile (hg19 NM_022493 c.482G>T) mutation in nuclear prelamin A recognition factor-like (NARFL) was identified. Ser161Ile mutation in NARFL conservation region was predicted to be deleterious and absent in 500 population controls and Exome Aggregation Consortium (ExAC) Database. And there was a dosage effect of the mutation on mRNA levels among family members and population controls, consistent with the instability of mutant mRNA in vitro. Accordingly, in lung tissue of the proband, NARFL protein expression was reduced but Fe3+ was overloaded with vascular endothelial growth factor (VEGF) overexpression. Furthermore, NARFL-knockdown cell lines demonstrated decreased activity of cytosolic aconitase, while NARFL-knockout zebrafish presented ectopic subintestinal vessels sprouts and upregulated VEGF. So we concluded that the Ser161Ile mutant induced NARFL deficiency and eventually diffuse PAVMs probably through VEGF pathway. In a word, we detected a functional mutation in NARFL, which might be the pathogenic gene in this pedigree.

The Phosphoinositide 3-Kinase Regulates Retrograde Trafficking of the Iron Permease CgFtr1 and Iron Homeostasis in Candida glabrata

The phosphoinositide 3-kinase (PI3K), which phosphorylates phosphatidylinositol and produces PI3P, has been implicated in protein trafficking, intracellular survival, and virulence in the pathogenic yeast Candida glabrata. Here, we demonstrate PI3-kinase (CgVps34) to be essential for maintenance of cellular iron homeostasis. We examine how CgVps34 regulates the fundamental process of iron acquisition, and underscore its function in vesicular trafficking as a central determinant. RNA sequencing analysis revealed iron homeostasis genes to be differentially expressed upon CgVps34 disruption. Consistently, the Cgvps34Δ mutant displayed growth attenuation in low- and high-iron media, increased intracellular iron content, elevated mitochondrial aconitase activity, impaired biofilm formation, and extenuated mouse organ colonization potential. Furthermore, we demonstrate for the first time that C. glabrata cells respond to iron limitation by expressing the iron permease CgFtr1 primarily on the cell membrane, and to iron excess via internalization of the plasma membrane-localized CgFtr1 to the vacuole. Our data show that CgVps34 is essential for the latter process. We also report that macrophage-internalized C. glabrata cells express CgFtr1 on the cell membrane indicative of an iron-restricted macrophage internal milieu, and Cgvps34Δ cells display better survival in iron-enriched medium-cultured macrophages. Overall, our data reveal the centrality of PI3K signaling in iron metabolism and host colonization.

Atypical microglial response to biodiesel exhaust in healthy and hypertensive rats

Accumulating evidence suggests a deleterious role for urban air pollution in central nervous system (CNS) diseases and neurodevelopmental disorders. Microglia, the resident innate immune cells and sentinels in the brain, are a common source of neuroinflammation and are implicated in air pollution-induced CNS effects. While renewable energy, such as soy-based biofuel, is of increasing public interest, there is little information on how soy biofuel may affect the brain, especially in people with preexisting disease conditions. To address this, male spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats were exposed to 100% Soy-based Biodiesel Exhaust (100SBDE; 0, 50, 150 and 500μg/m3) by inhalation, 4h/day for 4 weeks (5 days/week). Ionized calcium-binding adapter molecule-1 (IBA-1) staining of microglia in the substantia nigra revealed significant changes in morphology with 100SBDE exposure in rats from both genotypes, where SHR were less sensitive. Aconitase activity was inhibited in the frontal cortex and cerebellum of WKY rats exposed to 100SBDE. No consistent changes occurred in pro-inflammatory cytokine expression, nitrated protein, or arginase1 expression in brain regions from either rat strain exposed to 100SBDE. However, while IBA-1 mRNA expression was not modified, CX3CR1 mRNA expression was lower in the striatum of 100SBDE exposed rats regardless of genotype, suggesting a downregulation of the fractalkine receptor on microglia in this brain region. Together, these data indicate that while microglia are detecting and responding to 100SBDE exposure with changes in morphology, there is reduced expression of CX3CR1 regardless of genetic background and the activation response is atypical without traditional inflammatory markers of M1 or M2 activation in the brain.

A Small-Molecule Inhibitor of Iron-Sulfur Cluster Assembly Uncovers a Link between Virulence Regulation and Metabolism in Staphylococcus aureus

The rising problem of antimicrobial resistance in Staphylococcus aureus necessitates the discovery of novel therapeutic targets for small-molecule intervention. A major obstacle of drug discovery is identifying the target of molecules selected from high-throughput phenotypic assays. Here, we show that the toxicity of a small molecule termed '882 is dependent on the constitutive activity of the S.aureus virulence regulator SaeRS, uncovering a link between virulence factor production and energy generation. A series of genetic, physiological, and biochemical analyses reveal that '882 inhibits iron-sulfur (Fe-S) cluster assembly most likely through inhibition of the Suf complex, which synthesizes Fe-S clusters. In support of this, '882 supplementation results indecreased activity of the Fe-S cluster-dependent enzyme aconitase. Further information regarding the effects of '882 has deepened our understanding of virulence regulation and demonstrates the potential for small-molecule modulation of Fe-S cluster assembly in S.aureus and other pathogens.

Heterologous mitochondrial targeting sequences can deliver functional proteins into mitochondria

Mitochondrial Targeting Sequences (MTSs) are responsible for trafficking nuclear-encoded proteins into mitochondria. Once entering the mitochondria, the MTS is recognized and cleaved off. Some MTSs are long and undergo two-step processing, as in the case of the human frataxin (FXN) protein (80aa), implicated in Friedreich's ataxia (FA). Therefore, we chose the FXN protein to examine whether nuclear-encoded mitochondrial proteins can efficiently be targeted via a heterologous MTS (hMTS) and deliver a functional protein into mitochondria.We examined three hMTSs; that of citrate synthase (cs), lipoamide deydrogenase (LAD) and C6ORF66 (ORF), as classically MTS sequences, known to be removed by one-step processing, to deliver FXN into mitochondria, in the form of fusion proteins. We demonstrate that using hMTSs for delivering FXN results in the production of 4–5-fold larger amounts of the fusion proteins, and at 4–5-fold higher concentrations. Moreover, hMTSs delivered a functional FXN protein into the mitochondria even more efficiently than the native MTSfxn, as evidenced by the rescue of FA patients’ cells from oxidative stress; demonstrating a 18%–54% increase in cell survival; and a 13%-33% increase in ATP levels, as compared to the fusion protein carrying the native MTS. One fusion protein with MTScs increased aconitase activity within patients’ cells, by 400-fold.The implications form our studies are of vast importance for both basic and translational research of mitochondrial proteins as any mitochondrial protein can be delivered efficiently by an hMTS. Moreover, effective targeting of functional proteins is important for restoration of mitochondrial function and treatment of related disorders.

Use of antisense oligonucleotides to correct the splicing error in ISCU myopathy patient cell lines.

ISCU myopathy is an inherited disease that primarily affects individuals of northern Swedish descent who share a single point mutation in the fourth intron of the ISCU gene. The current study shows correction of specific phenotypes associated with disease following treatment with an antisense oligonucleotide (ASO) targeted to the site of the mutation. We have shown that ASO treatment diminished aberrant splicing and increased ISCU protein levels in both patient fibroblasts and patient myotubes in a concentration dependent fashion. Upon ASO treatment, levels of SDHB in patient myotubular cell lines increased to levels observed in control myotubular cell lines. Additionally, we have shown that both patient fibroblast and myotubular cell lines displayed an increase in complex II activity with a concomitant decrease in succinate levels in patient myotubular cell lines after ASO treatment. Mitochondrial and cytosolic aconitase activities increased significantly following ASO treatment in patient myotubes. The current study suggests that ASO treatment may serve as a viable approach to correcting ISCU myopathy in patients.