Risk Stratification in Pediatric Wolff-Parkinson-White: Practice Variation Among Pediatric Cardiologists and Electrophysiologists.

BACKGROUND: Published guidelines provide recommendations for risk stratification in pediatric Wolff-Parkinson-White (WPW). There are no data on provider concordance with these guidelines. We hypothesized that significant practice variation exists between pediatric cardiologists (PC) and electrophysiologists (EP). METHOD: The records of all patients, age 8 to 21 years, with a new ECG diagnosis of WPW between 1/1/2013 and 12/31/2018, from a single center, were retrospectively reviewed. Subjects were categorized on the basis of symptoms and resting ECG findings as one of the following: asymptomatic intermittent WPW, asymptomatic persistent WPW, or symptomatic WPW. The performance and results of diagnostic testing, including Holter monitor, event monitor, exercise stress test (EST), and electrophysiology study (EPS), were recorded. The primary outcome was concordance with published guidelines. A secondary outcome was documentation of a discussion of sudden cardiac death (SCD) risk. RESULTS: 615 patient encounters were analyzed in 231 patients with newly diagnosed WPW pattern on ECG (56% male; mean age at diagnosis 13.9 ± 2.5 years). EP were observed to have a significantly higher rate of guideline concordance than PC (95% vs. 71%, p < 0.001). There was significant practice variation between PC and EP in the documentation of a discussion of SCD risk: 96% in EP vs. 39% in PC (p < 0.001). CONCLUSION: Significant practice variation exists in the non-invasive and invasive risk stratification of pediatric WPW, with lower concordance to published guidelines amongst PC, when compared to EP. This report highlights the need to promote awareness of current WPW guidelines in the pediatric cardiology community at large.

Neonatal supraventricular tachycardia.

Supraventricular tachycardia (SVT) is one of the most common conditions requiring emergency cardiac care in neonates. Atrioventricular reentrant tachycardia utilizing an atrioventricular bypass tract is the most common form of SVT presenting in the neonatal period. There is high likelihood for spontaneous resolution for most of the common arrhythmia substrates in infancy. Pharmacological agents remain as the primary therapy for neonates.

Preincision Initiation of Dexmedetomidine Maximally Reduces the Risk of Junctional Ectopic Tachycardia in Children Undergoing Ventricular Septal Defect Repairs.

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.

Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid.

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.

Relationship between PCO2 and unfavorable outcome in infants with moderate-to-severe hypoxic ischemic encephalopathy.

BACKGROUND: Abnormal PCO2 is common in infants with hypoxic ischemic encephalopathy (HIE). The objective was to determine whether hypocapnia was independently associated with unfavorable outcome (death or severe neurodevelopmental disability at 18 mo) in infants with moderate-to-severe HIE. METHODS: This was a post hoc analysis of the CoolCap Study in which infants were randomized to head cooling or standard care. Blood gases were measured at prespecified times after randomization. PCO2 and follow-up data were available for 196 of 234 infants. Analyses were performed to investigate the relationship between hypocapnia in the first 72 h after randomization and unfavorable outcome. RESULTS: After adjusting for pH, amplitude-integrated electroencephalogram background and seizures, birth weight, Apgar score at 5 min, cooling status, and Sarnat stage, PCO2 was inversely associated with unfavorable outcome (P < 0.001). The probability of unfavorable outcome was 0.20 ± 0.1 (point estimate ± SE), 0.53 ± 0.23 and 0.89 ± 0.16 for a PCO2 of 40, 30, and 20 mm Hg respectively and was greater in infants with severe HIE than with moderate HIE. CONCLUSIONS: Hypocapnia in infants with moderate-to-severe HIE was independently associated with unfavorable outcome. Future studies of controlled normocapnia will be important.

Subcutaneous Implantable Cardioverter Defibrillators in Pediatrics and Congenital Heart Disease.

Subcutaneous implantable cardioverter defibrillators (S-ICDs) are being used with increased frequency in children and patients with congenital heart disease. Vascular access complexities, intracardiac shunts, and specific anatomies make these devices particularly appealing for some of these patients. Alternative screening, implantation, and programming techniques should be considered based on patient size, body habitus, anatomy, procedural history, and preference. Appropriate and inappropriate shock rates are generally comparable to those seen with transvenous devices. Complications such as infection can occur, although their severity is likely to be less than that seen with transvenous devices. Technical advances are likely to further broaden S-ICD applicability.

Delayed accumulation of intestinal coliform bacteria enhances life span and stress resistance in Caenorhabditis elegans fed respiratory deficient E. coli.

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.

Unincorporated iron pool is linked to oxidative stress and iron levels in Caenorhabditis elegans.

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.

Small-Molecule Mn Antioxidants in Caenorhabditis elegans and Deinococcus radiodurans Supplant MnSOD Enzymes during Aging and Irradiation.

Denham Harman's oxidative damage theory identifies superoxide (O2•-) radicals as central agents of aging and radiation injury, with Mn2+-dependent superoxide dismutase (MnSOD) as the principal O2•--scavenger. However, in the radiation-resistant nematode Caenorhabditis elegans, the mitochondrial antioxidant enzyme MnSOD is dispensable for longevity, and in the model bacterium Deinococcus radiodurans, it is dispensable for radiation resistance. Many radiation-resistant organisms accumulate small-molecule Mn2+-antioxidant complexes well-known for their catalytic ability to scavenge O2•-, along with MnSOD, as exemplified by D. radiodurans. Here, we report experiments that relate the MnSOD and Mn-antioxidant content to aging and oxidative stress resistances and which indicate that C. elegans, like D. radiodurans, may rely on Mn-antioxidant complexes as the primary defense against reactive oxygen species (ROS). Wild-type and ΔMnSOD D. radiodurans and C. elegans were monitored for gamma radiation sensitivities over their life spans while gauging Mn2+-antioxidant content by electron paramagnetic resonance (EPR) spectroscopy, a powerful new approach to determining the in vivo Mn-antioxidant content of cells as they age. As with D. radiodurans, MnSOD is dispensable for radiation survivability in C. elegans, which hyperaccumulates Mn-antioxidants exceptionally protective of proteins. Unexpectedly, ΔMnSOD mutants of both the nematodes and bacteria exhibited increased gamma radiation survival compared to the wild-type. In contrast, the loss of MnSOD renders radiation-resistant bacteria sensitive to atmospheric oxygen during desiccation. Our results support the concept that the disparate responses to oxidative stress are explained by the accumulation of Mn-antioxidant complexes which protect, complement, and can even supplant MnSOD. IMPORTANCE The current theory of cellular defense against oxidative damage identifies antioxidant enzymes as primary defenders against ROS, with MnSOD being the preeminent superoxide (O2•-) scavenger. However, MnSOD is shown to be dispensable both for radiation resistance and longevity in model organisms, the bacterium Deinococcus radiodurans and the nematode Caenorhabditis elegans. Measured by electron paramagnetic resonance (EPR) spectroscopy, small-molecule Mn-antioxidant content was shown to decline in unison with age-related decreases in cell proliferation and radioresistance, which again are independent of MnSOD presence. Most notably, the Mn-antioxidant content of C. elegans drops precipitously in the last third of its life span, which links with reports that the steady-state level of oxidized proteins increases exponentially during the last third of the life span in animals. This leads us to propose that global responses to oxidative stress must be understood through an extended theory that includes small-molecule Mn-antioxidants as potent O2•--scavengers that complement, and can even supplant, MnSOD.

Limitations of standard echocardiographic methods for quantification of right ventricular size and function in children and young adults.

OBJECTIVES: The aim of this study was to evaluate correlation of 2-dimensional (2D) echocardiographic assessment of right ventricular (RV) and left ventricular (LV) size and function with magnetic resonance imaging (MRI) in children and young adults. METHODS: Patients with repaired tetralogy of Fallot (n = 23) and those who had normal RV volumes (n = 13) and a normal ejection fraction (EF) by MRI constituted the study groups. Echocardiographic indices including the end-diastolic area (EDa), end-systolic area (ESa), fractional area change (FAC), tricuspid annular motion (TAM), RV basal diameter, and RV basal shortening fraction were compared with MRI ventricular volumes and the EF. Two echocardiographers qualitatively graded RV size and function. RESULTS: In both groups, neither the RV EDa nor the ESa correlated with MRI RV volumes. Only TAM correlated with the RV EF. Qualitative assessment of the RV showed poor interobserver agreement. The LV area and FAC correlated well with MRI data. CONCLUSIONS: In contrast to the LV, 2D echocardiographic indices of RV size and function, with the exception of TAM, do not correlate with MRI data.

Cardiac Relapse of Acute Lymphoblastic Leukemia Following Hematopoietic Stem Cell Transplantation: A Case Report and Review of Literature.

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.

Sudden Death in the Young: Information for the Primary Care Provider.

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.

An SCN1B Variant Affects Both Cardiac-Type (NaV1.5) and Brain-Type (NaV1.1) Sodium Currents and Contributes to Complex Concomitant Brain and Cardiac Disorders.

Voltage-gated sodium (NaV) channels are transmembrane proteins that initiate and propagate neuronal and cardiac action potentials. NaV channel ß subunits have been widely studied due to their modulatory role. Mice null for Scn1b, which encodes NaV ß1 and ß1b subunits, have defects in neuronal development and excitability, spontaneous generalized seizures, cardiac arrhythmias, and early mortality. A mutation in exon 3 of SCN1B, c.308A>T leading to ß1_p.D103V and ß1b_p.D103V, was previously found in a patient with a history of proarrhythmic conditions with progressive atrial standstill as well as cognitive and motor deficits accompanying structural brain abnormalities. We investigated whether ß1 or ß1b subunits carrying this mutation affect NaV1.5 and/or NaV1.1 currents using a whole cell patch-clamp technique in tsA201 cells. We observed a decrease in sodium current density in cells co-expressing NaV1.5 or NaV1.1 and ß1D103V compared to ß1WT. Interestingly, ß1bD103V did not affect NaV1.1 sodium current density but induced a positive shift in the voltage dependence of inactivation and a faster recovery from inactivation compared to ß1bWT. The ß1bD103V isoform did not affect NaV1.5 current properties. Although the SCN1B_c.308A>T mutation may not be the sole cause of the patient's symptoms, we observed a clear loss of function in both cardiac and brain sodium channels. Our results suggest that the mutant ß1 and ß1b subunits play a fundamental role in the observed electrical dysfunction.

Only one of a wide assortment of manganese-containing SOD mimicking compounds rescues the slow aerobic growth phenotypes of both Escherichia coli and Saccharomyces cerevisiae strains lacking superoxide dismutase enzymes.

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.

Manganous ion supplementation accelerates wild type development, enhances stress resistance, and rescues the life span of a short-lived Caenorhabditis elegans mutant.

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.

Cognitive impairments following cranial irradiation can be mitigated by treatment with a tropomyosin receptor kinase B agonist.

Brain radiotherapy is frequently used successfully to treat brain tumors. However, radiotherapy is often associated with declines in short-term and long-term memory, learning ability, and verbal fluency. We previously identified a downregulation of the brain-derived neurotrophic factor (BDNF) following cranial irradiation in experimental animals. In the present study, we investigated whether targeting the BDNF high affinity receptor, tropomysin receptor kinase B (TrkB), could mitigate radiation-induced cognitive deficits. After irradiation, chronic treatment with a small molecule TrkB agonist, 7,8-dihydroxyflavone (DHF) in mice led to enhanced activation of TrkB and its downstream targets ERK and AKT, both important factors in neuronal development. DHF treatment significantly restored spatial, contextual, and working memory, and the positive effects persisted for at least 3months after completion of the treatment. Consistent with preservation of cognitive functions, chronic DHF treatment mitigated radiation-induced suppression of hippocampal neurogenesis. Spine density and major components of the excitatory synapses, including glutamate receptors and postsynaptic density protein 95 (PSD-95), were also maintained at normal levels by DHF treatment after irradiation. Taken together, our results show that chronic treatment with DHF after irradiation significantly mitigates radiation-induced cognitive defects. This is achieved most likely by preservation of hippocampal neurogenesis and synaptic plasticity.

Diagnosis and Acute Management of Tachyarrhythmias in Children.

Tachyarrhythmias form a significant majority of the cardiac emergencies encountered in the pediatric age group. While the initial management often depends on the hemodynamic status of the patient at presentation, a systematic approach that follows the stabilization of the patient with the tools available at bedside, specifically the surface electrocardiogram often results in optimal treatment response that is tailored towards the mechanism and severity of the arrhythmia. The goal of this review is to provide the pediatric cardiologist with such an approach based on clinical history and analysis of the electrocardiogram (ECG). Illustrative examples are used to explain this approach towards the more common tachyarrhythmias. A general overview of the initial anti-arrhythmic interventions will be provided.

Competition between lithium and magnesium ions for the G-protein transducin in the guanosine 5'-diphosphate bound conformation.

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(+)/Mg(2+) 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 Mg(2+), the apparent Li(+) binding constant decreased due to Li(+)/Mg(2+) competition. Whereas for SROS the competition mechanism was monophasic, for G(t), the competition was biphasic, suggesting that in G(t), Li(+)/Mg(2+) competition occurred with different affinities for Mg(2+) in two types of Mg(2+) binding sites. Moreover, as [Li(+)] increased, the fluorescence excitation spectra of both ROS-T and G(t) were blue shifted, indicating an increase in free [Mg(2+)] compatible with Li(+) displacement of Mg(2+) from two low affinity Mg(2+) binding sites of G(t). G(t) release from ROS-T membrane was also inhibited by Li(+) addition. In summary, we found evidence of Li(+)/Mg(2+) competition in G(t)-containing preparations.

Analysis of the transcriptome in hyperoxic lung injury and sex-specific alterations in gene expression.

Exposure to high concentration of oxygen (hyperoxia) leads to lung injury in experimental animal models and plays a role in the pathogenesis of diseases such as Acute Respiratory Distress Syndrome (ARDS) and Bronchopulmonary dysplasia (BPD) in humans. The mechanisms responsible for sex differences in the susceptibility towards hyperoxic lung injury remain largely unknown. The major goal of this study was to characterize the changes in the pulmonary transcriptome following hyperoxia exposure and further elucidate the sex-specific changes. Male and female (8-10 wk) wild type (WT) (C57BL/6J) mice were exposed to hyperoxia (FiO2>0.95) and gene expression in lung tissues was studied at 48 h. A combination of fold change ≥1.4 and false discovery rate (FDR)<5% was used to define differentially expressed genes (DEGs). Overrepresentation of gene ontology terms representing biological processes and signaling pathway impact analysis (SPIA) was performed. Comparison of DEG profiles identified 327 genes unique to females, 585 unique to males and 1882 common genes. The major new findings of this study are the identification of new candidate genes of interest and the sex-specific transcriptomic changes in hyperoxic lung injury. We also identified DEGs involved in signaling pathways like MAP kinase and NF-kappa B which may explain the differences in sex-specific susceptibility to hyperoxic lung injury. These findings highlight changes in the pulmonary transcriptome and sex-specific differences in hyperoxic lung injury, and suggest new pathways, whose components could serve as sex-specific biomarkers and possible therapeutic targets for acute lung injury (ALI)/acute respiratory distress (ARDS) in humans.

Restoring de novo coenzyme Q biosynthesis in Caenorhabditis elegans coq-3 mutants yields profound rescue compared to exogenous coenzyme Q supplementation.

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 Q(10). Here we show that the Q(9) 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 Q(9) 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.