Screen for New Antimicrobial Natural Products from the NCI Program for Natural Product Discovery Prefractionated Extract Library.

The continuing emergence of antibiotic-resistant microbes highlights the need for the identification of new chemotypes with antimicrobial activity. One of the most prolific sources of antimicrobial molecules has been the systematic screening of natural product samples. The National Institute of Allergy and Infectious Diseases and the National Cancer Institute here report a large screen of 326,656 partially purified natural product fractions against a panel of four microbial pathogens, resulting in the identification of >3000 fractions with antifungal and/or antibacterial activity. A small sample of these active fractions was further purified and the chemical structures responsible for the antimicrobial activity were elucidated. The proof-of-concept study identified many different chemotypes, several of which have not previously been reported to have antimicrobial activity. The results show that there remain many unidentified antibiotic compounds from nature.

Modulation of the cardiac sodium channel NaV1.5 peak and late currents by NAD+ precursors.

RATIONALE: The cardiac sodium channel NaV1.5, encoded by SCN5A, produces the rapidly inactivating depolarizing current INa that is responsible for the initiation and propagation of the cardiac action potential. Acquired and inherited dysfunction of NaV1.5 results in either decreased peak INa or increased residual late INa (INa,L), leading to tachy/bradyarrhythmias and sudden cardiac death. Previous studies have shown that increased cellular NAD+ and NAD+/NADH ratio increase INa through suppression of mitochondrial reactive oxygen species and PKC-mediated NaV1.5 phosphorylation. In addition, NAD+-dependent deacetylation of NaV1.5 at K1479 by Sirtuin 1 increases NaV1.5 membrane trafficking and INa. The role of NAD+ precursors in modulating INa remains unknown. OBJECTIVE: To determine whether and by which mechanisms the NAD+ precursors nicotinamide riboside (NR) and nicotinamide (NAM) affect peak INa and INa,Lin vitro and cardiac electrophysiology in vivo. METHODS AND RESULTS: The effects of NAD+ precursors on the NAD+ metabolome and electrophysiology were studied using HEK293 cells expressing wild-type and mutant NaV1.5, rat neonatal cardiomyocytes (RNCMs), and mice. NR increased INa in HEK293 cells expressing NaV1.5 (500 µM: 51 ± 18%, p = .02, 5 mM: 59 ± 22%, p = .03) and RNCMs (500 µM: 60 ± 26%, p = .02, 5 mM: 74 ± 39%, p = .03) while reducing INa,L at the higher concentration (RNCMs, 5 mM: -45 ± 11%, p = .04). NR (5 mM) decreased NaV1.5 K1479 acetylation but increased INa in HEK293 cells expressing a mutant form of NaV1.5 with disruption of the acetylation site (NaV1.5-K1479A). Disruption of the PKC phosphorylation site abolished the effect of NR on INa. Furthermore, NAM (5 mM) had no effect on INa in RNCMs or in HEK293 cells expressing wild-type NaV1.5, but increased INa in HEK293 cells expressing NaV1.5-K1479A. Dietary supplementation with NR for 10-12 weeks decreased QTc in C57BL/6 J mice (0.35% NR: -4.9 ± 2.0%, p = .14; 1.0% NR: -9.5 ± 2.8%, p = .01). CONCLUSIONS: NAD+ precursors differentially regulate NaV1.5 via multiple mechanisms. NR increases INa, decreases INa,L, and warrants further investigation as a potential therapy for arrhythmic disorders caused by NaV1.5 deficiency and/or dysfunction.

High-energy external defibrillation and transcutaneous pacing during MRI: feasibility and safety.

BACKGROUND: Rapid application of external defibrillation, a crucial first-line therapy for ventricular fibrillation and cardiac arrest, is currently unavailable in the setting of magnetic resonance imaging (MRI), raising concerns about patient safety during MRI tests and MRI-guided procedures, particularly in patients with cardiovascular diseases. The objective of this study was to examine the feasibility and safety of defibrillation/pacing for the entire range of clinically useful shock energies inside the MRI bore and during scans, using defibrillation/pacing outside the magnet as a control. METHODS: Experiments were conducted using a commercial defibrillator (LIFEPAK 20, Physio-Control, Redmond, Washington, USA) with a custom high-voltage, twisted-pair cable with two mounted resonant floating radiofrequency traps to reduce emission from the defibrillator and the MRI scanner. A total of 18 high-energy (200-360 J) defibrillation experiments were conducted in six swine on a 1.5 T MRI scanner outside the magnet bore, inside the bore, and during scanning, using adult and pediatric defibrillation pads. Defibrillation was followed by cardiac pacing (with capture) in a subset of two animals. Monitored signals included: high-fidelity temperature (0.01 °C, 10 samples/sec) under the pads and 12-lead electrocardiogram (ECG) using an MRI-compatible ECG system. RESULTS: Defibrillation/pacing was successful in all experiments. Temperature was higher during defibrillation inside the bore and during scanning compared with outside the bore, but the differences were small (ΔT: 0.5 and 0.7 °C, p = 0.01 and 0.04, respectively). During scans, temperature after defibrillation tended to be higher for pediatric vs. adult pads (p = 0.08). MR-image quality (signal-to-noise ratio) decreased by ~ 10% when the defibrillator was turned on. CONCLUSIONS: Our study demonstrates the feasibility and safety of in-bore defibrillation for the full range of defibrillation energies used in clinical practice, as well as of transcutaneous cardiac pacing inside the MRI bore. Methods for Improving MR-image quality in the presence of a working defibrillator require further study.

Sirtuin 1 regulates cardiac electrical activity by deacetylating the cardiac sodium channel.

The voltage-gated cardiac Na+ channel (Nav1.5), encoded by the SCN5A gene, conducts the inward depolarizing cardiac Na+ current (INa) and is vital for normal cardiac electrical activity. Inherited loss-of-function mutations in SCN5A lead to defects in the generation and conduction of the cardiac electrical impulse and are associated with various arrhythmia phenotypes. Here we show that sirtuin 1 deacetylase (Sirt1) deacetylates Nav1.5 at lysine 1479 (K1479) and stimulates INa via lysine-deacetylation-mediated trafficking of Nav1.5 to the plasma membrane. Cardiac Sirt1 deficiency in mice induces hyperacetylation of K1479 in Nav1.5, decreases expression of Nav1.5 on the cardiomyocyte membrane, reduces INa and leads to cardiac conduction abnormalities and premature death owing to arrhythmia. The arrhythmic phenotype of cardiac-Sirt1-deficient mice recapitulated human cardiac arrhythmias resulting from loss of function of Nav1.5. Increased Sirt1 activity or expression results in decreased lysine acetylation of Nav1.5, which promotes the trafficking of Nav1.5 to the plasma membrane and stimulation of INa. As compared to wild-type Nav1.5, Nav1.5 with K1479 mutated to a nonacetylatable residue increases peak INa and is not regulated by Sirt1, whereas Nav1.5 with K1479 mutated to mimic acetylation decreases INa. Nav1.5 is hyperacetylated on K1479 in the hearts of patients with cardiomyopathy and clinical conduction disease. Thus, Sirt1, by deacetylating Nav1.5, plays an essential part in the regulation of INa and cardiac electrical activity.

Elevated neonatal insulin-like growth factor I is associated with fetal hypertrophic cardiomyopathy in diabetic women.

OBJECTIVE: We sought to determine if fetal hypertrophic cardiomyopathy (HCM) or cardiac dysfunction is associated with elevated maternal or neonatal insulin-like growth factor (IGF)-I levels in women with diabetes. STUDY DESIGN: In a prospective cohort study, fetal echocardiogram findings at 36 weeks' gestation in women with pregestational diabetes mellitus were compared to those in women without diabetes mellitus. HCM was defined as septal or free wall thickness ≥5 mm and cardiac dysfunction as a modified myocardial performance index ≥0.43. Cord serum IGF-I levels at delivery were measured with enzyme-linked immunosorbent assay. Neonates with abnormal fetal echocardiogram were followed up until resolution or 6 months of life. RESULTS: In all, 75 participants completed fetal echocardiography (55 diabetics and 20 controls). In the diabetic group, 33 of 55 (60%) had abnormal fetal echocardiograms with cardiac dysfunction in 21 of 55 (38.2%) and HCM in 8 of 55 (14.5%) and both in 4 of 55 (7.3%). At 6 months of age, 1 of 12 (8%) had persistent HCM. None in the comparison group had abnormal findings. There were no significant clinical differences in those diabetic women with normal vs abnormal fetal echocardiograms. However, among diabetic women, mean neonatal IGF-I was significantly higher in fetuses with HCM (80 ± 16 ng/mL) as compared to those without HCM (61 ± 18 ng/mL), (P < .001). CONCLUSION: Elevated neonatal IGF-I appears to be associated with fetal HCM in fetuses of diabetic women.

Corticosterone administration does not affect timing of breeding in Florida scrub-jays (Aphelocoma coerulescens).

Providing supplemental food to Florida scrub-jays (Aphelocoma coerulescens) causes a reliable advance in clutch initiation of 1 to 2 weeks. In some years, supplemental food appeared to not only advance laying date but also decrease baseline concentrations of corticosterone (CORT) relative to controls. The coincidence of low CORT levels and early breeding led us to hypothesize that CORT serves to communicate information about environmental conditions to the hypothalamic-pituitary-gonadal (HPG) axis, which ultimately influences the timing of breeding. To test this hypothesis, we administered small oral doses of CORT three times each day to female breeders that were provisioned with supplemental food. We compared clutch initiation dates of the CORT-dosed females to females with supplementation but no exogenous CORT and to females with neither CORT nor supplemental food. CORT administration had a strong temporary effect on circulating CORT concentrations but clutch initiation did not differ between the two groups of supplemented birds, both of which laid eggs approximately 10 days earlier than nonsupplemented birds. Furthermore, during the year of our study we found no reduction in baseline CORT concentrations in our undosed supplemental groups, as had been observed in past studies.

Role of selective leptin resistance in diet-induced obesity hypertension.

Leptin is an adipocyte-derived hormone that plays a key role in the regulation of body weight through its actions on appetite and metabolism. Leptin also increases sympathetic nerve activity (SNA) and blood pressure. We tested the hypothesis that diet-induced obesity is associated with resistance to the metabolic actions of leptin but preservation of its renal SNA and arterial pressure effects, leading to hypertension. Mice were fed a high-fat diet for 10 weeks to induce moderate obesity. The decrease in food intake and body weight induced by intraperitoneal or intracerebroventricular leptin was significantly attenuated in the obese mice. Regional SNA responses to leptin were differentially altered in diet-induced obese mice. Renal SNA response to leptin was preserved, whereas lumbar and brown adipose tissue SNA responses were attenuated in obese mice. Radiotelemetric arterial pressure was approximately 10 mmHg higher in obese mice. Furthermore, the increase in arterial pressure in response to long-term (12 days) leptin treatment was preserved in obese mice. Thus, mice with diet-induced obesity exhibit circulating hyperleptinemia and resistance to the metabolic actions of leptin. However, there is preservation of the renal sympathetic and arterial pressure responses to leptin, which represent a potential mechanism for the adverse cardiovascular consequences of obesity.

Hypothalamic PI3K and MAPK differentially mediate regional sympathetic activation to insulin.

The action of insulin in the central nervous system produces sympathetic nervous system activation (also called sympathoactivation), although the neuronal intracellular mechanisms that mediate this are unclear. We hypothesized that PI3K and MAPK, the major pathways involved in insulin receptor signaling, mediate sympathetic nerve responses to insulin. Intracerebroventricular administration of insulin in rat increased multifiber sympathetic nerve activity to the hindlimb, brown adipose tissue (BAT), adrenal gland, and kidney. Ex vivo biochemical studies of mediobasal hypothalamic tissue revealed that insulin stimulated the association of insulin receptor substrate-1 with the p85alpha subunit of PI3K and also tyrosine phosphorylation of p42 and p44 subunits of MAPK in the hypothalamus. In order to determine whether PI3K and/or MAPK were involved in insulin-mediated sympathoactivation, we tested the effect of specific inhibitors of PI3K (LY294002 and wortmannin) and MAPK (PD98059 and U0126) on regional sympathetic responses to insulin. Interestingly, regional sympathoactivation to insulin was differentially affected by blockade of PI3K and MAPK. Inhibition of PI3K specifically blocked insulin-induced sympathoactivation to the hindlimb, while inhibition of MAPK specifically blocked insulin-induced sympathoactivation to BAT. Sympathoactivation to corticotrophin-releasing factor, however, was not affected by inhibition of PI3K and MAPK. These data demonstrate that PI3K and MAPK are specific and regionally selective mediators of the action of insulin on the sympathetic nervous system.