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During several decades, there has been a meteoric rise in the development and use of cutting-edge technology. The Wireless Sensor Network (WSN) is a groundbreaking innovation that relies on a vast network of individual sensor nodes. The sensor nodes in the network are responsible for collecting data and uploading it to the cloud. When networks with little resources are deployed harshly and without regulation, security risks occur. Since the rate at which new information is being generated is increasing at an exponential rate, WSN communication has become the most challenging and complex aspect of the field. Therefore, WSNs are insecure because of this. With so much riding on WSN applications, accuracy in replies is paramount. Technology that can swiftly and continually analyse internet data streams is essential for spotting breaches and assaults. Without categorization, it is hard to simultaneously reduce processing time while maintaining a high level of detection accuracy. This paper proposed using a Multi-Layer Perceptron (MLP) to enhance the classification accuracy of a system. The proposed method utilises a feed-forward ANN model to generate a mapping for the training and testing datasets using backpropagation. Experiments are performed to determine how well the proposed MLP works. Then, the results are compared to those obtained by using the Hoeffding adaptive tree method and the Restricted Boltzmann Machine-based Clustered-Introduction Detection System. The proposed MLP achieves 98% accuracy, which is higher than the 96.33% achieved by the RBMC-IDS and the 97% accuracy achieved by the Hoeffding adaptive tree.

Simple Summary We present a case of a pediatric patient with symptomatic leukemia involving the heart without bone marrow involvement. The case is followed by a literature review of advances in leukemia treatment including immunotherapy, CAR T-cell therapy, improvement in the treatment of graft-versus-host disease, and the applicability of these treatments to leukemia involving areas outside of the bone marrow. We also explore the feasibility of treatments such as CAR T-cell therapy and blinatumomab and the associated risks for treating patients with leukemia infiltrating the heart. Abstract Isolated extramedullary relapse of acute lymphoblastic leukemia (ALL) occurs in soft tissues and various organs outside the testis and central nervous system. Treatments such as hematopoietic stem cell transplantation and more novel modalities such as immunotherapy have eradicated ALL at extramedullary sites. In some instances, survival times for relapsed ALL at these sites are longer than those for relapsed disease involving only the bone marrow. Isolated relapse of ALL in the myocardium is rare, especially in children, making diagnosis and treatment of it difficult. More recent treatment options such as chimeric antigen receptor T-cell therapy carry a high risk of cytokine release syndrome and associated risk of worsening cardiac function. Herein we present the case of an 11-year-old boy who presented with relapsed symptomatic B-cell ALL in the myocardium following allogeneic hematopoietic stem cell transplantation. This is an unusual presentation of relapsed ALL and this case demonstrates the associated challenges in its diagnosis and treatment. The case report is followed by a literature review of the advances in treatment of pediatric leukemia and their application to extramedullary relapse of this disease in particular.

There are multiple conditions that can make children prone to having a sudden cardiac arrest (SCA) or sudden cardiac death (SCD). Efforts have been made by multiple organizations to screen children for cardiac conditions, but the emphasis has been on screening before athletic competition. This article is an update of the previous American Academy of Pediatrics policy statement of 2012 that addresses prevention of SCA and SCD. This update includes a comprehensive review of conditions that should prompt more attention and cardiology evaluation. The role of the primary care provider is of paramount importance in the evaluation of children, particularly as they enter middle school or junior high. There is discussion about whether screening should find any cardiac condition or just those that are associated with SCA and SCD. This update reviews the 4 main screening questions that are recommended, not just for athletes, but for all children. There is also discussion about how to handle post-SCA and SCD situations as well as discussion about genetic testing. It is the goal of this policy statement update to provide the primary care provider more assistance in how to screen for life-threatening conditions, regardless of athletic status.

Paroxysmal tachyarrhythmias (TA) are challenging to diagnose in children given their difficulty in characterizing their varying symptomatology as well as frequently sporadic nature of presentation. The purpose of this study is to determine the diagnostic yield of mobile cardiac telemetry (MCT) as a

Coq9 is a polypeptide subunit in a mitochondrial multi-subunit complex, termed the CoQ-synthome, required for biosynthesis of coenzyme Q (ubiquinone or Q). Deletion of COQ9 results in dissociation of the CoQ-synthome, but over-expression of Coq8 putative kinase stabilizes the CoQ-synthome in the coq9 null mutant and leads to the accumulation of two nitrogen-containing Q intermediates, imino-demethoxy-Q6 (IDMQ6) and 3-hexaprenyl-4-aminophenol (4-AP) when para-aminobenzoic acid (pABA) is provided as a ring precursor. To investigate whether Coq9 is responsible for deamination steps in Q biosynthesis, we utilized the yeast coq5-5 point mutant. The yeast coq5-5 point mutant is defective in the C-methyltransferase step of Q biosynthesis but retains normal steady-state levels of the Coq5 polypeptide. Here, we show that when high amounts of 13C6-pABA are provided, the coq5-5 mutant accumulates both 13C6-imino-demethyl-demethoxy-Q6 (13C6-IDDMQ6) and 13C6-demethyl-demethoxy-Q6 (13C6-DDMQ6). Deletion of COQ9 in the yeast coq5-5 mutant along with Coq8 over-expression and 13C6- pABA labeling leads to the absence of 13C6-DDMQ6, and the nitrogen-containing intermediates 13C6-4-AP and 13C6-IDDMQ6 persist. We describe a coq9 temperature-sensitive mutant and show that at the non-permissive temperature, steady-state polypeptide levels of Coq9-ts19 increased, while Coq4, Coq5, Coq6, and Coq7 decreased. The coq9-ts19 mutant had decreased Q6 content and increased levels of nitrogen-containing intermediates. These findings identify Coq9 as a multi-functional protein that is required for the function of Coq6 and Coq7 hydroxylases, for removal of the nitrogen substituent from pABA-derived Q intermediates, and is an essential component of the CoQ synthome.

Objective To evaluate whether initiation of dexmedetomidine (DEX) infusion before surgical incision and cardiopulmonary bypass (CPB) versus initiation after CPB had an impact on the incidence of junctional ectopic tachycardia (JET). Design Retrospective cohort study. Setting Single tertiary-care cardiac center. Participants Children undergoing cardiopulmonary bypass for repair of congenital heart disease involving ventricular septal defects between January 2010 and February 2013. Interventions None. Measurements and Main Results One hundred thirty-four patients undergoing ventricular septal defect closure were included in the final analysis. Of the 99 patients (74%) exposed to DEX, intraoperative initiation was performed in 73 (pre-CPB, n = 39 patients [29%]; intraoperative post-CPB initiation, n = 34 patients [25%]), and postoperative initiation was performed on arrival to the intensive care unit (ICU) in 26 patients (19%). In 71 of the 73 patients, infusions that were initiated intraoperatively were continued in the postoperative period for up to the first 12 hours. Postoperative JET was observed in 22 of the 134 patients (15%). Of the 99 patients exposed to DEX in the perioperative period, JET was observed in 8 patients (11%). Of the 35 patients not exposed to any DEX, JET was observed in 12 patients (34%). Analysis was performed using DEX exposure and timing as predictor variables. Multivariable analysis modeled with DEX exposure as a predictor variable showed that when initiated preincision and continued through the postoperative period, DEX was associated with significant reduction in postoperative JET (odds ratio [OR] 0.09, 95% confidence interval [CI] 0.02-0.37, p = 0.002). Exposure to DEX in the postoperative period alone did not result in suppression of JET (OR 0.5, 95% CI 0.11-2.17, p = 0.366). When modeled by using timing of DEX initiation as the predictive variable, preincision initiation of DEX infusion resulted in significantly greater suppression of JET (OR 0.04, 95% CI 0.002-0.28, p = 0.006) compared with initiation intraoperatively after CPB (OR 0.16, 95% CI 0.03-0.71, p = 0.024) or on arrival to the ICU (OR 0.504, CI 0.105-2.171, p = 0.365). Use of DEX exclusively in the postoperative period did not demonstrate any significant benefit in reducing JET (OR 0.506, 95% CI 0.106-2.17, p = 0.366). Conclusions Preincision initiation of DEX and its continued use during the immediate postoperative period are significantly associated with reduced risk of JET after congenital heart surgeries involving repair of ventricular septal defect.

The family of superoxide dismutases (SODs) have an almost ubiquitous presence in living organisms. Their importance in maintaining normal cellular functions and fate decisions has been demonstrated in a large number of studies since their discovery in 1968. Studies of pathological conditions resulting from naturally occurring SOD mutations in human populations or from altered SOD levels created in animal models further highlighted the critical roles of SODs in maintaining the redox balance for normal tissue functions. The production of adult-born neurons in the hippocampus is important for the neuronal plasticity of learning and memory, and yet the process is exquisitely sensitive to changes in the redox balance in its microenvironment. Consequently, mutant mice deficient in CuZnSOD or MnSOD showed altered progenitor cell differentiation towards the astroglial, instead of the neuronal, lineage, while EC-SOD deficient mice exhibited a significant reduction in the production of newborn neurons. Similarly, ionizing radiation, even at low doses, led to persistent perturbation of the tissue redox environment and suppression of hippocampal neurogenesis. The defects in hippocampal neurogenesis resulting from EC-SOD deficiency or ionizing irradiation also correlated with impairments in hippocampal-dependent functions of learning and memory. To correct the persistent redox imbalance following irradiation, enhanced EC-SOD levels in mature hippocampal neurons were achieved by transgenic approach. The transgenic mice were able to minimize irradiation-induced suppression of hippocampal neurogenesis and the associated cognitive defects, suggesting that SOD-based antioxidant supplementation may be an efficacious therapeutic approach to CNS defects following ionizing radiation therapy. The new generation Mn-containing porphyrins had been demonstrated to possess high SOD-like activities, and some of them were able to cross the blood brain barrier. Their radioprotective capacity had been demonstrated in a number of tissues in radiation animal models. Application of Mn porphyrins for the preservation of hippocampal neurogenesis and cognitive functions in experimental models will increase their potential relevance towards human radiation therapy of the CNS.

Free radicals or reactive oxygen species (ROS) are relatively short-lived and are difficult to measure directly; so indirect methods have been explored for measuring these transient species. One technique that has been developed using Escherichia coli and Saccharomyces cerevisiae systems, relies on a connection between elevated superoxide levels and the build-up of a high-spin form of iron (Fe(III)) that is detectable by electron paramagnetic resonance (EPR) spectroscopy at g = 4.3. This form of iron is referred to as "free" iron. EPR signals at g = 4.3 are commonly encountered in biological samples owing to mononuclear high-spin (S = 5/2) Fe(III) ions in sites of low symmetry. Unincorporated iron in this study refers to this high-spin Fe(III) that is captured by desferrioxamine which is detected by EPR at g value of 4.3. Previously, we published an adaptation of Fe(III) EPR methodology that was developed for Caenorhabditis elegans, a multi-cellular organism. In the current study, we have systematically characterized various factors that modulate this unincorporated iron pool. Our results demonstrate that the unincorporated iron as monitored by Fe(III) EPR at g = 4.3 increased under conditions that were known to elevate steady-state ROS levels in vivo, including: paraquat treatment, hydrogen peroxide exposure, heat shock treatment, or exposure to higher growth temperature. Besides the exogenous inducers of oxidative stress, physiological aging, which is associated with elevated ROS and ROS-mediated macromolecular damage, also caused a build-up of this iron. In addition, increased iron availability increased the unincorporated iron pool as well as generalized oxidative stress. Overall, unincorporated iron increased under conditions of oxidative stress with no change in total iron levels. However, when total iron levels increased in vivo, an increase in both the pool of unincorporated iron and oxidative stress was observed suggesting that the status of the unincorporated iron pool is linked to oxidative stress and iron levels.

Oxidative stress, caused by free radicals within the body, has been associated with the process of aging and many human diseases. Because free radicals, in particular superoxide, are difficult to measure, an alternative indirect method for measuring oxidative stress levels has been used successfully in Escherichia coli and yeast. This method is based on a proposed connection between elevated superoxide levels and release of iron from solvent-exposed [4Fe-4S] enzyme clusters that eventually leads to an increase in hydroxyl radical production. In past studies using bacteria and yeast, a positive correlation was found between superoxide production or oxidative stress due to superoxide within the organism and electron paramagnetic resonance (EPR) detectable "free" iron levels. In the current study, we have developed a reliable and efficient method for measuring "free" iron levels in Caenorhabditis elegans using low-temperature Fe(III) EPR at g=4.3. This method uses synchronized worm cultures grown on plates that are homogenized and treated with desferrioxamine, an Fe(III) chelator, prior to packing the EPR tube. Homogenization was found not to alter "free" iron levels, whereas desferrioxamine treatment significantly raised these levels, indicating the presence of both Fe(II) and Fe(III) in the "free" iron pool. The correlation between free radical levels and the observed "free" iron levels was examined by using heat stress and paraquat treatment. The intensity of the Fe(III) EPR signal, and thus the concentration of the "free" iron pool, varied with the treatments that altered radical levels without changing the total iron levels. This study provides the groundwork needed to uncover the correlation among oxidative stress, "free" iron levels, and longevity in C. elegans.

Yeasts lacking copper-zinc superoxide dismutase (sod1Delta) exhibit a broad range of phenotypes, many of which can be rescued by growth in the presence of high levels of ionic manganese. We undertook a comprehensive survey of the effects of manganese on wild-type and sod1Delta yeasts and found that 5 mM Mn2+ rescued all known growth-related phenotypes, such as slow growth in air, temperature sensitivity, specific amino acid auxotrophies, no growth in high oxygen, poor growth in nonfermentable carbon sources, and decreased stationary-phase survival. Iron-related phenotypes-elevated electron paramagnetic resonance detectable ("free") iron, decreased aconitase activity, and fragmenting vacuoles-as well as zinc sensitivity were also rescued. The activity of manganese superoxide dismutase remained constant or was reduced when the yeasts were grown in the presence of MnCl2, indicating that induction of this alternative superoxide dismutase is not the explanation. In contrast to MnCl2 treatment, addition of two manganese-containing superoxide dismutase mimetic compounds to the growth medium did not provide any rescue of sod1Delta yeast growth but rather had an sod1Delta-selective inhibitory effect at micromolar concentrations. Mechanisms by which ionic manganese can effect this rescue, while the mimetic compounds do not, are discussed.

Saccharomyces cerevisiae lacking Cu,Zn superoxide dismutase (SOD1) show several metabolic defects including aerobic blockages in methionine and lysine biosynthesis. We have previously shown that mutations in genes implicated in the formation of iron-sulfur clusters, designated seo (suppressors of endogenous oxidation), reverse the oxygen-dependent methionine and lysine auxotrophies of a sod1Delta strain. We now report the surprising finding that seo mutants do not reduce oxidative damage as shown by the lack of reduction of EPR-detectable "free" iron, which is characteristic of sod1Delta mutants. In fact, they exhibit increased oxidative damage as evidenced by increased accumulation of protein carbonyls. The seo class of mutants overaccumulates mitochondrial iron, and this iron accumulation is critical for suppression of the sod1Delta biosynthetic defects. Blocking overaccumulation of mitochondrial iron abolished the ability of the seo mutants to suppress the sod1Delta auxotrophies. By contrast, increasing the mitochondrial iron content of sod1Delta yeast using high copy MMT1, which encodes a mitochondrial iron transporter, was sufficient to mimic the seo mutants. Our studies indicated that suppression of the sod1Delta methionine auxotrophy was dependent on the pentose phosphate pathway, which is a major source of NADPH production. By comparison, the sod1Delta lysine auxotrophy appears to be reversed in the seo mutants by increased expression of genes in the lysine biosynthetic pathway, perhaps through sensing of mitochondrial damage by the retrograde response.

Li+ is the most effective drug used to treat bipolar disorder; however, its exact mechanism of action has yet to be elucidated. One hypothesis is that Li+ competes with Mg2+ for the Mg2+ binding sites on guanine-nucleotide binding proteins (G-proteins). Using 7Li T1 relaxation measurements and fluorescence spectroscopy with the Mg2+ fluorophore furaptra, we detected Li+/Mg2+ competition in three preparations: the purified G-protein transducin (Gt), stripped rod outer segment membranes (SROS), and SROS with purified Gt reattached (ROS-T). When purified ROS-T, SROS or transducin were titrated with Li+ in the presence of fixed amounts of Mg2+, the apparent Li+ binding constant decreased due to Li+/Mg2+ competition. Whereas for SROS the competition mechanism was monophasic, for Gt, the competition was biphasic, suggesting that in Gt, Li+/Mg2+ competition occurred with different affinities for Mg2+ in two types of Mg2+ binding sites. Moreover, as [Li+] increased, the fluorescence excitation spectra of both ROS-T and Gt were blue shifted, indicating an increase in free [Mg2+] compatible with Li+ displacement of Mg2+ from two low affinity Mg2+ binding sites of Gt. Gt release from ROS-T membrane was also inhibited by Li+ addition. In summary, we found evidence of Li+/Mg2+ competition in Gt-containing preparations. http://www.sciencedirect.com/science/article/B6TGG-4BJ2538-2/1/c5cc8bd5992b6629aba06705e0861119

Although lithium salts have been used in the treatment and prophylaxis of manic-depressive or bipolar patients for 50 years, the mechanism of the pharmacologic action of Li + is unknown. Based on activity studies of inhibitory and stimulatory guanine-binding (G) proteins in rat cortical membranes, it was proposed that Li + inhibition of G-proteins may account for its pharmacologic action. We used the purified α subunit of the recombinant inhibitory G-protein, rG iα1 , and found that Li + at therapeutic levels significantly inhibited the formation of the GDP.AlF − 4 .rG iα1 complex. Because our studies were conducted with a purified, metal-reconstitutedG-protein rather than with cell membrane suspensions, our Li + inhibition results lend additional support to the G-protein hypothesis for Li + action.

Coenzyme Q (ubiquinone or Q) is an essential lipid component of the mitochondrial electron transport chain. In Caenorhabditis elegans Q biosynthesis involves at least nine steps, including the hydroxylation of the hydroquinone ring by CLK-1 and two O-methylation steps mediated by COQ-3. We characterize two C. elegans coq-3 deletion mutants, and show that while each has defects in Q synthesis, their phenotypes are distinct. First generation homozygous coq-3(ok506) mutants are fertile when fed the standard lab diet of Q-replete OP50 Escherichia coli, but their second generation homozygous progeny does not reproduce. In contrast, the coq-3(qm188) deletion mutant remains sterile when fed Q-replete OP50. Quantitative PCR analyses suggest that the longer qm188 deletion may alter expression of the flanking nuo-3 and gdi-1 genes, located 5′ and 3′, respectively of coq-3 within an operon. We surmise that variable expression of nuo-3, a subunit of complex I, or of gdi-1, a guanine nucleotide dissociation inhibitor, may act in combination with defects in Q biosynthesis to produce a more severe phenotype. The phenotypes of both coq-3 mutants are more drastic as compared to the C. elegans clk-1 mutants. When fed OP50, clk-1 mutants reproduce for many generations, but show reduced fertility, slow behaviors, and enhanced life span. The coq-3 and clk-1 mutants all show arrested development and are sterile when fed the Q-deficient E. coli strain GD1 (harboring a mutation in the ubiG gene). However, unlike clk-1 mutant worms, neither coq-3 mutant strain responded to dietary supplementation with purified exogenous Q10. Here we show that the Q9 content can be determined in lipid extracts from just 200 individual worms, enabling the determination of Q content in the coq-3 mutants unable to reproduce. An extra-chromosomal array expressing wild-type C. elegans coq-3 rescued fertility of both coq-3 mutants and partially restored steady-state levels of COQ-3 polypeptide and Q9 content, indicating that primary defect in both is limited to coq-3. The limited response of the coq-3 mutants to dietary supplementation with Q provides a powerful model to probe the effectiveness of exogenous Q supplementation as compared to restoration of de novo Q biosynthesis.

We investigated the mechanism of competition between Li+ and Mg2+ in Li(+)-loaded human red blood cells (RBCs) by making 7Li and 31P NMR and fluorescence measurements. We used 7Li NMR relaxation times to probe Li+ binding to the human RBC membrane and ATP; an increase in Mg2+ concentration caused an increase in both 7Li T1 and T2 values in packed Li(+)-loaded RBCs, in suspensions of Li(+)-loaded RBC ghosts, in suspensions of Li(+)-containing RBC membrane, and in aqueous solutions of ATP, indicating competition between Li+ and Mg2+ for binding sites in the membrane and ATP. We found that increasing concentrations of either Li+ or Mg2+ in the presence of human RBC membrane caused an increase in the 31P NMR chemical shift anisotropy parameter, which describes the observed axially symmetric powder pattern, indicating metal ion binding to the phosphate groups in the membrane. Competition between Li+ and Mg2+ for phosphate groups in ATP and in the RBC membrane was also observed by both fluorescence measurements and 31P NMR spectroscopy at low temperature. The ratio of the stoichiometric binding constants of Mg2+ to Li+ to the RBC membrane was approximately 20; the ratio of the conditional binding constants in the presence of a free intracellular ATP concentration of 0.2 mM was approximately 4, indicating that Li+ competes for approximately 20% of the Mg(2+)-binding sites in the RBC membrane. Our results indicate that, regardless of the spectroscopic method used, Li+ competes with Mg2+ for phosphate groups in both ATP and the RBC membrane; the extent of metal ion competition for the phosphate head groups of the phospholipids in the RBC membrane is enhanced by the presence of ATP. Competition between Li+ and Mg2+ for anionic phospholipids or Mg(2+)-activated proteins present in cell membranes may constitute the basis of a general molecular mechanism for Li+ action in human tissues.

Somatic growth pattern in infants undergoing staged palliation for hypoplastic left heart syndrome (Norwood procedure [NP], stage 2 palliation [S2P], and Fontan procedure [FP]) during transition toward a more energy efficient series circulation is not well understood.We sought to determine growth pattern in these infants and factors influencing it.Patients who underwent NP since 2001 and survived at least 1 year after S2P were followed until FP or death/heart transplantation (n = 46). Weight for age z-scores (WAZ) were measured prior to NP; at initial discharge; prior to S2P; at four time periods after S2P; and prior to FP. Impact of gender, race, era of NP, anatomic subtype, NP shunt type, gastrostomy, home-surveillance program (daily weights and pulse oximetry), and interstage oxygen saturation on WAZ was evaluated.Repeated measures anova showed a significant change in WAZ across time periods (P0.0005), with a significant decline from Pre-NP to Pre-S2P (P0.0005) and steady increase from Pre-S2P to Pre-FP (P= 0.016). None of the factors evaluated had a significant effect on this growth pattern. Length of hospital stay after NP was negatively correlated with WAZ at discharge (P= 0.001), but not for other time periods. Intervals from discharge to S2P and NP to S2P interval did not correlate with WAZ.Somatic growth is significantly impaired after NP but recovers steadily following S2P, as the patient transitions toward separated series circulation. Earlier S2P may lead to earlier resumption of normal growth in this critical period of early infancy.

Human behavior can change through external influence. However, internal influences, such as individual perceptions, are more powerful in triggering long-term action, ultimately allowing for substantial changes in behavioral patterns. Applying the aforementioned idea to farming practices, the following study seeks to understand the possible association between risk perception and agro-chemical use patterns between conventional and organic farms, the former using chemical pesticides and fertilizers.

Background. In the past decade, many advances in the care of patients undergoing the Norwood procedure (NP) have been reported, but management remains nonstandardized at many institutions. We studied the impact of a standardized management protocol for neonates undergoing NP. Methods. Care of NP patients has been protocol-driven at our institution since 2005, with routine use of regional low flow perfusion; near infrared spectroscopy; phenoxybenzamine with cardiopulmonary bypass; delayed sternal closure; peritoneal drainage; gastrostomy tubes; postoperative vocal cord assessment; and a home surveillance program of daily weight and oxygen saturation measurement. Patients undergoing NP from 2001 to 2004 (n = 40, group 1), in whom these interventions were only selectively employed, were retrospectively compared with those receiving standardized management from 2005 to 2007 (n = 40, group 2), with endpoints of survival in-hospital and to stage 2 palliation (S2P). Effect of protocol elements on outcome was evaluated by univariate and multivariate analyses. Results. Hospital survival (95% vs. 70%, P= .003) and survival to S2P (85% vs. 58%, P= .006) was better in group 2. By univariate analysis, regional low flow perfusion, gastrostomy usage, and near infrared spectroscopy were associated with improved hospital and survival to S2P. In multivariable analysis, gastrostomy usage was associated with improved hospital survival (P= .027) and survival to S2P (P= .049), while our home surveillance program was a predictor of survival to S2P (P= .016). Conclusion. Protocol-driven management of NP patients was associated with better hospital survival and survival to S2P. Among protocol elements, gastrostomy usage was linked to both improved hospital survival and survival to S2P. Home surveillance was associated with increased survival to S2P.

A variety of manganese-containing coordination compounds, frequently termed superoxide dismutase (SOD) mimics, have been reported to have SOD activity in vitro and to be effective at improving conditions related to increased oxidative stress in multicellular organisms. We tested the effectiveness of several of these compounds in substituting for authentic SOD enzymes in two simple systems--the prokaryote Escherichia coli and the single-celled eukaryote, Saccharomyces cerevisiae--where strains are available that completely lack cytoplasmic SOD activity and are thus significantly impaired in their ability to grow aerobically. Most of the compounds tested, including Euk-8 and Euk-134, manganese salen derivatives developed by Eukarion; M40403, a manganese complex of a bis(cyclohexylpyridine)-substituted macrocyclic ligand developed by Metaphore; and several manganese porphyrin derivatives, were ineffective in both systems. Only the manganese tetrapyridyl porphyrin complex MnTM-2-PyP and two close relatives were effective in rescuing aerobic growth of E. coli lacking SOD, and, in the case of sod1Delta yeast, only MnTM-2-PyP itself was fully effective. Surprisingly, several compounds reported to be beneficial in other in vivo model systems (Euk-8, Euk-134, M40403) were actually toxic to these organisms lacking SOD, although they had no effect on the wild-type parent strains. Our results suggest the possibility that the beneficial effects of some of the so-called "SOD mimic drugs" may be due to some property other than in vivo superoxide dismutase activity.

Relative to iron and copper we know very little about the cellular roles of manganese. Some studies claim that manganese acts as a radical scavenger in unicellular organisms, while there have been other reports that manganese causes Parkinson's disease-like syndrome, DNA fragmentation, and interferes with cellular energy production. The goal of this study was to uncover if manganese has any free radical scavenging properties in the complex multicellular organism, Caenorhabditis elegans. We measured internal manganese in supplemented worms using inductively coupled plasma mass spectrometry (ICP-MS) and the data obtained suggest that manganese supplemented to the growth medium is taken up by the worms. We found that manganese did not appear to be toxic as supplementation did not negatively effect development or fertility. In fact, supplementation at higher levels accelerated development and increased total fertility of wild type worms by 16%. Manganese-supplemented wild type worms were found to be thermotolerant and, under certain conditions, long-lived. In addition, the oxidatively challenged C. elegans strain mev-1's short life span was significantly increased after manganese supplementation. Although manganese appears to be beneficial to C. elegans, the mode of action remains unclear. Manganese may work directly as a free radical scavenger, as it has been postulated to do so in unicellular organisms, or may work indirectly by up regulating several protective factors.

The LYS7 gene in Saccharomyces cerevisiae encodes a protein (yCCS) that delivers copper to the active site of copper-zinc superoxide dismutase (CuZn-SOD, a product of the SOD1 gene). In yeast lacking Lys7 (lys7Delta), the SOD1 polypeptide is present but inactive. Mutants lacking the SOD1 polypeptide (sod1Delta) and lys7Delta yeast show very similar phenotypes, namely poor growth in air and aerobic auxotrophies for lysine and methionine. Here, we demonstrate certain phenotypic differences between these strains: 1) lys7Delta cells are slightly less sensitive to paraquat than sod1Delta cells, 2) EPR-detectable or "free" iron is dramatically elevated in sod1Delta mutants but not in lys7Delta yeast, and 3) although sod1Delta mutants show increased sensitivity to extracellular zinc, the lys7Delta strain is as resistant as wild type. To restore the SOD catalytic activity but not the zinc-binding capability of the SOD1 polypeptide, we overexpressed Mn-SOD from Bacillus stearothermophilus in the cytoplasm of sod1Delta yeast. Paraquat resistance was restored to wild-type levels, but zinc was not. Conversely, expression of a mutant CuZn-SOD that binds zinc but has no SOD activity (H46C) restored zinc resistance but not paraquat resistance. Taken together, these results strongly suggest that CuZn-SOD, in addition to its antioxidant properties, plays a role in zinc homeostasis.

The copper metallochaperone Cox17 is proposed to shuttle Cu(I) ions to the mitochondrion for the assembly of cytochromec oxidase. The Cu(I) ions are liganded by cysteinyl thiolates. Mutational analysis on the yeast Cox17 reveals three of the seven cysteinyl residues to be critical for Cox17 function, and these three residues are present in a Cys-Cys-Xaa-Cys sequence motif. Single substitution of any of these three cysteines with serines results in a nonfunctional cytochrome oxidase complex. Cells harboring such a mutation fail to grow on nonfermentable carbon sources and have no cytochrome c oxidase activity in isolated mitochondria. Wild-type Cox17 purified as untagged protein binds three Cu(I) ions/molecule. Mutant proteins lacking only one of these critical Cys residues retain the ability to bind three Cu(I) ions and are imported within the mitochondria. In contrast, Cox17 molecules with a double Cys → Ser mutation exhibit no Cu(I) binding but are still localized to the mitochondria. Thus, mitochondrial uptake of Cox17 is not restricted to the Cu(I) conformer of Cox17. COX17 was originally cloned by virtue of complementation of a mutant containing a nonfunctional Cys → Tyr substitution at codon 57. The mutant C57Y Cox17 fails to accumulate within the mitochondria but retains the ability to bind three Cu(I) ions. A C57S Cox17 variant is functional, and a quadruple Cox17 mutant with C16S/C36S/C47S/C57S substitutions binds three Cu(I) ions. Thus, only three cysteinyl residues are important for the ligation of three Cu(I) ions. A novel mode of Cu(I) binding is predicted.

The Zap1 transcriptional activator of Saccharomyces cerevisiae controls zinc homeostasis. Zap1 induces target gene expression in zinc-limited cells and is repressed by high zinc. One such target gene is ZAP1 itself. In this report, we examine how zinc regulates Zap1 function. First, we show that transcriptional autoregulation of Zap1 is a minor component of zinc responsiveness; most regulation of Zap1 activity occurs post-translationally. Secondly, nuclear localization of Zap1 does not change in response to zinc, suggesting that zinc regulates DNA binding and/or activation domain function. To understand how Zap1 responds to zinc, we performed a functional dissection of the protein. Zap1 contains two activation domains. DNA-binding activity is conferred by five C-terminal C(2)H(2) zinc fingers and each finger is required for high-affinity DNA binding. The zinc-responsive domain of Zap1 also maps to the C-terminal zinc fingers. Furthermore, mutations that disrupt some of these fingers cause constitutive activity of a bifunctional Gal4 DNA-binding domain-Zap1 fusion protein. These results demonstrate a novel function of Zap1 zinc fingers in zinc sensing as well as DNA binding.

A current hypothesis explaining the toxicity of superoxide anion in vivo is that it oxidizes exposed [4Fe-4S] clusters in certain vulnerable enzymes causing release of iron and enzyme inactivation. The resulting increased levels of "free iron" catalyze deleterious oxidative reactions in the cell. In this study, we used low temperature Fe(III) electron paramagnetic resonance (EPR) spectroscopy to monitor iron status in whole cells of the unicellular eukaryote, Saccharomyces cerevisiae. The experimental protocol involved treatment of the cells with desferrioxamine, a cell-permeant, Fe(III)-specific chelator, to promote oxidation of all of the "free iron" to the Fe(III) state wherein it is EPR-detectable. Using this method, a small amount of EPR-detectable iron was detected in the wild-type strain, whereas significantly elevated levels were found in strains lacking CuZn-superoxide dismutase (CuZn-SOD) (sod1 delta), Mn-SOD (sod2 delta), or both SODs, throughout their growth but particularly in stationary phase. The accumulation was suppressed by expression of wild-type human CuZn-SOD (in the sod1 delta mutant), by pmr1, a genetic suppressor of the sod delta mutant phenotype (in the sod1 delta sod2 delta double knockout strain), and by anaerobic growth. In wild-type cells, an increase in the EPR-detectable iron pool could be induced by treatment with paraquat, a redox-cycling drug that generates superoxide. Cells that were not pretreated with desferrioxamine had Fe(III) EPR signals that were equally as strong as those from treated cells, indicating that "free iron" accumulated in the ferric form in our strains in vivo. Our results indicate a relationship between superoxide stress and iron handling and support the above hypothesis for superoxide-related oxidative damage.

Mac1 is a metalloregulatory protein that regulates expression of the high affinity copper transport system in the yeast Saccharomyces cerevisiae. Under conditions of high copper concentration, Mac1 represses transcription of genes coding for copper transport proteins. Mac1 binds to DNA sequences called copper response elements (CuREs), which have the consensus sequence 5'-TTTGC(T/G)C(A/G)-3'. Mac1 contains two zinc binding sites, a copper binding site, and the sequence motif RGRP, which has been found in other proteins to mediate binding to the minor groove of A/T-rich sequences in DNA. We have used hydroxyl radical footprinting, missing nucleoside, and methylation interference experiments to investigate the structure of the complex of the DNA binding domain of Mac1 (called here Mac1(t)) with the two CuRE sites found in the yeast CTR1 promoter. We conclude from these experiments that Mac1(t) binds in a modular fashion to DNA, with its RGRP AT-hook motif interacting with the TTT sequence at the 5' end of the CTR1 CuRE site, and with another DNA-binding module(s) binding in the adjacent major groove in the GCTCA sequence.

Mac1 from Saccharomyces cerevisiae activates transcription of genes, including CTR1 in copper-deficient cells. N-terminal fusions of Mac1 with the herpes simplex VP16 activation domain were used to show that residues 1-159 in Mac1 constitute the minimal DNA binding domain. Mac1-(1-159) purified from Escherichia coli contains two bound Zn(II) ions. Electrophoretic mobility shift assays showed direct and specific binding by Mac1-(1-159) to a DNA duplex containing the copper-responsive element TTTGCTCA. The DNA binding affinity of Mac1-(1-159) for a duplex containing a single promoter element or an inverted repeat was 5 nM for the 1:1 complex. The N-terminal 40-residue segment of Mac1 is homologous to the DNA binding zinc module found in the copper-activated transcription factors Ace1 and Amt1. A MAC1 mutation yielding a Cys11 --> Tyr substitution at the first candidate zinc ligand position relative to Ace1 resulted in a loss of in vivo function. Two TTTGCTCA promoter elements are necessary for efficient Mac1-mediated transcriptional activation. The elements appear to function synergistically. Increasing the number of elements yields more than additive enhancements in CTR1 expression.

Assembly of functional cytochrome oxidase in yeast requires Cox17, which has been postulated to deliver copper ions to the mitochondrion for insertion into the enzyme. This role for Cox17 is supported by the observation that it binds copper as a binuclear cuprous−thiolate cluster. X-ray absorption spectroscopy, together with UV−visible absorption and emission spectroscopy, indicates the presence of bound cuprous ions, trigonally coordinated by thiolate ligands. Analysis of the EXAFS shows three Cu−S bonds at 2.26 A, plus a short Cu−Cu distance of 2.7 A, indicating a binuclear cluster in Cox17. The cuprous−thiolate cluster in Cox17 is substantially more labile than structurally related clusters in metallothioneins.

Evidence for competition between Li(+) and Na(+) for binding sites of human unsealed and cytoskeleton-depleted human red blood cell (csdRBC) membranes was obtained from the effect of added Li(+) upon the (23)Na double quantum filtered (DQF) and triple quantum filtered (TQF) NMR signals of Na(+)-containing red blood cell (RBC) membrane suspensions. We found that, at low ionic strength, the observed quenching effect of Li(+) on the (23)Na TQF and DQF signal intensity probed Li(+)/Na(+) competition for isotropic binding sites only. Membrane cytoskeleton depletion significantly decreased the isotropic signal intensity, strongly affecting the binding of Na(+) to isotropic membrane sites, but had no effect on Li(+)/Na(+) competition for those sites. Through the observed (23)Na DQF NMR spectra, which allow probing of both isotropic and anisotropic Na(+) motion, we found anisotropic membrane binding sites for Na(+) when the total ionic strength was higher than 40 mM. This is a consequence of ionic strength effects on the conformation of the cytoskeleton, in particular on the dimer-tetramer equilibrium of spectrin. The determinant involvement of the cytoskeleton in the anisotropy of Na(+) motion at the membrane surface was demonstrated by the isotropy of the DQF spectra of csdRBC membranes even at high ionic strength. Li(+) addition initially quenched the isotropic signal the most, indicating preferential Li(+)/Na(+) competition for the isotropic membrane sites. High ionic strength also increased the intensity of the anisotropic signal, due to its effect on the restructuring of the membrane cytoskeleton. Further Li(+) addition competed with Na(+) for those sites, quenching the anisotropic signal. (7)Li T(1) relaxation data for Li(+)-containing suspensions of unsealed and csdRBC membranes, in the absence and presence of Na(+) at low ionic strength, showed that cytoskeleton depletion does not affect the affinity of Na(+) for the RBC membrane, but increases the affinity of Li(+) by 50%. This clearly indicates that cytoskeleton depletion favors Li(+) relative to Na(+) binding, and thus Li(+)/Na(+) competition for its isotropic sites. Thus, this relaxation technique proves to be very sensitive to alkali metal binding to the membrane, detecting a more pronounced steric hindrance effect of the cytoskeleton network to binding of the larger hydrated Li(+) ion to the membrane phosphate groups.

Background Studies with the nematode model Caenorhabditis elegans have identified conserved biochemical pathways that act to modulate life span. Life span can also be influenced by the composition of the intestinal microbiome, and C. elegans life span can be dramatically influenced by its diet of Escherichia coli. Although C. elegans is typically fed the standard OP50 strain of E. coli, nematodes fed E. coli strains rendered respiratory deficient, either due to a lack coenzyme Q or the absence of ATP synthase, show significant life span extension. Here we explore the mechanisms accounting for the enhanced nematode life span in response to these diets. Results The intestinal load of E. coli was monitored by determination of worm-associated colony forming units (cfu/worm or coliform counts) as a function of age. The presence of GFP-expressing E. coli in the worm intestine was also monitored by fluorescence microscopy. Worms fed the standard OP50 E. coli strain have high cfu and GFP-labeled bacteria in their guts at the L4 larval stage, and show saturated coliform counts by day five of adulthood. In contrast, nematodes fed diets of respiratory deficient E. coli lacking coenzyme Q lived significantly longer and failed to accumulate bacteria within the lumen at early ages. Animals fed bacteria deficient in complex V showed intermediate coliform numbers and were not quite as long-lived. The results indicate that respiratory deficient Q-less E. coli are effectively degraded in the early adult worm, either at the pharynx or within the intestine, and do not accumulate in the intestinal tract until day ten of adulthood. Conclusions The findings of this study suggest that the nematodes fed the respiratory deficient E. coli diet live longer because the delay in bacterial colonization of the gut subjects the worms to less stress compared to worms fed the OP50 E. coli diet. This work suggests that bacterial respiration can act as a virulence factor, influencing the ability of bacteria to colonize and subsequently harm the animal host. Respiratory deficient bacteria may pose a useful model for probing probiotic relationships within the gut microbiome in higher organisms.