The Hybrid Antibiotic Thiomarinol A Overcomes Intrinsic Resistance in Escherichia coli Using a Privileged Dithiolopyrrolone Moiety.

An impermeable outer membrane and multidrug efflux pumps work in concert to provide Gram-negative bacteria with intrinsic resistance against many antibiotics. These resistance mechanisms reduce the intracellular concentrations of antibiotics and render them ineffective. The natural product thiomarinol A combines holothin, a dithiolopyrrolone antibiotic, with marinolic acid A, a close analogue of mupirocin. The hybridity of thiomarinol A converts the mupirocin scaffold from inhibiting Gram-positive bacteria to inhibiting both Gram-positive and -negative bacteria. We found that thiomarinol A accumulates significantly more than mupirocin within the Gram-negative bacterium Escherichia coli, likely contributing to its broad-spectrum activity. Antibiotic susceptibility testing of E. coli mutants reveals that thiomarinol A overcomes the intrinsic resistance mechanisms that render mupirocin inactive. Structure-activity relationship studies suggest that the dithiolopyrrolone is a privileged moiety for improving the accumulation and antibiotic activity of the mupirocin scaffold without compromising binding to isoleucyl-tRNA synthetase. These studies also highlight that accumulation is required but not sufficient for antibiotic activity. Our work reveals a role of the dithiolopyrrolone moiety in overcoming intrinsic mupirocin resistance in E. coli and provides a starting point for designing dual-acting and high-accumulating hybrid antibiotics.

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.

Impact of Neuroeffector Adrenergic Receptor Polymorphisms on Incident Ventricular Fibrillation During Acute Myocardial Ischemia.

Background Cardiac adrenergic receptor gene polymorphisms have the potential to influence risk of developing ventricular fibrillation (VF) during ST-segment-elevation myocardial infarction, but no previous study has comprehensively investigated those most likely to alter norepinephrine release, signal transduction, or biased signaling. Methods and Results In a case-control study, we recruited 953 patients with ST-segment-elevation myocardial infarction without previous cardiac history, 477 with primary VF, and 476 controls without VF, and genotyped them for ADRB1 Arg389Gly and Ser49Gly, ADRB2 Gln27Glu and Gly16Arg, and ADRA2C Ins322-325Del. Within each minor allele-containing genotype, haplotype, or 2-genotype combination, patients with incident VF were compared with non-VF controls by odds ratios (OR) of variant frequencies referenced against major allele homozygotes. Of 156 investigated genetic constructs, 19 (12.2%) exhibited significantly (P<0.05) reduced association with incident VF, and none was associated with increased VF risk except for ADRB1 Gly389 homozygotes in the subset of patients not receiving ß-blockers. ADRB1 Gly49 carriers (prevalence 23.0%) had an OR (95% CI) of 0.70 (0.49-0.98), and the ADRA2C 322-325 deletion (Del) carriers (prevalence 13.5%) had an OR of 0.61 (0.39-0.94). When present in genotype combinations (8 each), both ADRB1 Gly49 carriers (OR, 0.67 [0.56-0.80]) and ADRA2C Del carriers (OR, 0.57 [0.45- 0.71]) were associated with reduced VF risk. Conclusions In ST-segment-elevation myocardial infarction, the adrenergic receptor minor alleles ADRB1 Gly49, whose encoded receptor undergoes enhanced agonist-mediated internalization and ß-arrestin interactions leading to cardioprotective biased signaling, and ADRA2C Del322-325, whose receptor causes disinhibition of norepinephrine release, are associated with a lower incidence of VF. Registration URL: https://clinicaltrials.gov; Unique identifier: NCT00859300.

Enzymatic Synthesis of Diverse Heterocycles by a Noncanonical Nonribosomal Peptide Synthetase.

Nonribosomal peptide synthetases (NRPSs) are typically multimodular enzymes that assemble amino acids or carboxylic acids into complex natural products. Here, we characterize a monomodular NRPS, PvfC, encoded by the Pseudomonas virulence factor (pvf) gene cluster that is essential for virulence and signaling in different bacterial species. PvfC exhibits a unique adenylation-thiolation-reductase (ATR) domain architecture that is understudied in bacteria. We show that the activity of PvfC is essential in the production of seven leucine-derived heterocyclic natural products, including two pyrazines, a pyrazinone, and a rare disubstituted imidazole, as well as three pyrazine N-oxides that require an additional N-oxygenation step. Mechanistic studies reveal that PvfC, without a canonical peptide-forming domain, makes a dipeptide aldehyde intermediate en route to both the pyrazinone and imidazole. Our work identifies a novel biosynthetic route for the production of pyrazinones, an emerging class of signaling molecules and virulence factors. Our discovery also showcases the ability of monomodular NRPSs to generate amino acid- and dipeptide-aldehydes that lead to diverse natural products. The diversity-prone biosynthesis by the pvf-encoded enzymes sets the stage for further understanding the functions of pvf in bacterial cell-to-cell signaling.

Pseudomonas Virulence Factor Pathway Synthesizes Autoinducers That Regulate the Secretome of a Pathogen.

Cell-to-cell communication via chemical signals is an essential mechanism that pathogenic bacteria use to coordinate group behaviors and promote virulence. The Pseudomonas virulence factor (pvf) gene cluster is distributed in more than 500 strains of proteobacteria including both plant and human pathogens. The pvf cluster has been implicated in the production of signaling molecules important for virulence; however, the regulatory impact of these signaling molecules on virulence had not been elucidated. Using the insect pathogen Pseudomonas entomophila L48 as a model, we demonstrated that pvf-encoded biosynthetic enzymes produce PVF autoinducers that regulate the expression of pvf genes and a gene encoding the toxin monalysin via quorum sensing. In addition, PVF autoinducers regulate the expression of nearly 200 secreted and membrane proteins, including toxins, motility proteins, and components of the type VI secretion system, which play key roles in bacterial virulence, colonization, and competition with other microbes. Deletion of pvf also altered the secondary metabolome. Six major compounds upregulated by PVF autoinducers were isolated and structurally characterized, including three insecticidal 3-indolyl oxazoles, the labradorins, and three antimicrobial pyrrolizidine alkaloids, the pyreudiones. The signaling properties of PVF autoinducers and their wide-ranging regulatory effects indicate multifaceted roles of PVF in controlling cell physiology and promoting virulence. The broad genome distribution of pvf suggests that PVF-mediated signaling is relevant to many bacteria of agricultural and biomedical significance.

In Vitro Reconstitution Reveals a Central Role for the N-Oxygenase PvfB in (Dihydro)pyrazine-N-oxide and Valdiazen Biosynthesis.

The Pseudomonas virulence factor (pvf) operon is essential for the biosynthesis of two very different natural product scaffolds: the (dihydro)pyrazine-N-oxides and the diazeniumdiolate, valdiazen. PvfB is a member of the non-heme diiron N-oxygenase enzyme family that commonly convert anilines to their nitroaromatic counterparts. In contrast, we show that PvfB catalyzes N-oxygenation of the α-amine of valine, first to the hydroxylamine and then the nitroso, while linked to the carrier protein of PvfC. PvfB modification of PvfC-tethered valine was observed directly by protein NMR spectroscopy, establishing the intermediacy of the hydroxylamine. This work reveals a central role for PvfB in the biosynthesis of (dihydro)pyrazine-N-oxides and valdiazen.

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.

Specificity of Nonribosomal Peptide Synthetases in the Biosynthesis of the Pseudomonas virulence factor.

The Pseudomonas virulence factor (pvf) biosynthetic operon has been implicated in bacterial virulence and signaling. We identified 308 bacterial strains containing pvf homologues that likely produce signaling molecules with distinct structures and biological activities. Several homologues of the nonribosomal peptide synthetase (NRPS), PvfC, were biochemically characterized and shown to activate l-Val or l-Leu. The amino acid selectivity of PvfC and its homologues likely direct pvf signaling activity. We explored the natural diversity of the active site residues present in 92% of the adenylation domains of PvfC homologues and identified key residues for substrate selection and catalysis. Sequence similarity network (SSN) analysis revealed grouping of PvfC homologues that harbor the same active site residues and activate the same amino acids. Our work identified PvfC as a gatekeeper for the structure and bioactivity of the pvf-produced signaling molecules. The combination of active site residue identification and SSN analysis can improve the prediction of aliphatic amino acid substrates for NRPS adenylation domains.

Magnetic Resonance Imaging of Contracting Ultrathin Cardiac Tissue.

OBJECTIVE: Integrating cardiac-tissue patches into the beating heart and evaluating the long-term effects of such integration on cardiac contractility are two challenges in an emerging field of regenerative medicine. This pilot study presents tools for the imaging of contracting multicellular cardiac tissue constructs (MTCs) in vitro and demonstrates the feasibility of tracking the early development of strand geometry and contractions in ultrathin strands and layers of cardiac tissue using CINE MRI. APPROACH: Cultured, ultrathin (~50-100-micron) MTCs of rat neonatal cardiomyocytes were plated in rectangular cell chambers (4.5 × 2.0 cm) with and without ultrathin, carbon EP electrodes embedded in the floor of the cell chamber. Two-dimensional, steady-state free precession (SSFP) CINE MRI, cell microscopy, and tissue photography were performed on Days 5-9 of cell development. Potential confounders and MRI artifacts were evaluated using non-contracting cardiac tissues and cell-free chambers filled with the cell-culture medium. MAIN RESULTS: Synchronized contractions formed by Day 7; individual contracting tissue strands became identifiable by Day 9. The global patterns and details of the strand geometry and movement patterns in the SSFP images were in excellent agreement with microscopic and photographic images. No synchronized movement was identifiable by either microscopy or CINE MRI in the non-contracting MTCs or the cell-free medium. The EP recordings revealed well-defined depolarization and repolarization waveforms; the imaging artifacts generated by the carbon electrodes were small. SIGNIFICANCE: This pilot study demonstrates the feasibility of imaging cardiac-strand patterns and contractile activity in ultrathin, two-dimensional cardiac tissue in commonly used clinical scanners.

Discovery of (Dihydro)pyrazine N-Oxides via Genome Mining in Pseudomonas.

Overexpression of the Pseudomonas virulence factor ( pvf) biosynthetic operon led to the identification of a family of pyrazine N-oxides (PNOs), including a novel dihydropyrazine N,N'-dioxide (dPNO) metabolite. The nonribosomal peptide synthetase responsible for production of (d)PNOs was characterized, and a biosynthetic pathway for (d)PNOs was proposed. This work highlights the unique chemistry catalyzed by pvf-encoded enzymes and sets the stage for bioactivity studies of the metabolites produced by the virulence pathway.

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.

Periprostatic adipose tissue from obese prostate cancer patients promotes tumor and endothelial cell proliferation: a functional and MR imaging pilot study.

BACKGROUND: Obesity, particularly visceral adiposity, confers a worse prognosis for prostate cancer (PCa) patients, and increasing periprostatic adipose (PPA) tissue thickness or density is positively associated with more aggressive disease. However, the cellular mechanism of this activity remains unclear. Therefore, in this pilot study, we assessed the functional activity of PPA tissue secretions and established a biochemical profile of PPA as compared to subcutaneous adipose (SQA) tissues from lean, overweight and obese PCa patients. METHODS: Adipose tissues were collected from PCa patients undergoing surgical prostate removal. Tissues were analyzed by histologic and magnetic resonance (MR) techniques. Explant tissue culture secretions were used in proliferation assays on PCa and endothelial cells. RESULTS: PPA secretions obtained from obese patients were significantly more pro-proliferative in both PCa and endothelial cells as compared to PPA obtained from lean or overweight men and SQA tissues. Consistent with this, PPA microvessel density was increased, and the T2 relaxation time was decreased, compared to SQA tissues, and we observed a modest, inverse correlation between the T2 and tumor stage. Moreover, the ratio of unsaturated to saturated fatty acids, obtained using MR spectroscopy, showed a modest, inverse correlation with Gleason score. CONCLUSIONS: These pilot data show that PPA stimulates PCa cell proliferation and angiogenesis and that obesity intensifies this activity, thus generating a mechanistic hypothesis to explain the worse prognosis observed in obese PCa patients. Our pilot study also shows that MR technology may be useful in further elucidating the relationship between obesity and PCa progression.

A study of best practices for clinical documentation system implementation.

Nurses are key leaders and team members for the implementation of clinical documentation systems. This paper reviews key success factors during implementation of clinical documentation systems to overcome traditional barriers, ensure commitment to universal goals and facilitate the use of technology for patient safety and continuous quality improvement efforts. Top level management support, particularly from nursing executives, is vital.

2008 HIMSS Survey results: best practices in implementing nursing/interdisciplinary documentation systems.

Health care organizations are increasingly using computer systems to support nursing care documentation; however, processes used to deploy such systems are widely varied. The purpose of this survey was to understand current practices related to implementation of computerized nursing and interdisciplinary documentation systems with the goal to establish best practice guidelines. In Spring 2007, members from the Healthcare Information Management and Systems Society Nursing Informatics Working Group interviewed a sample of 15 hospitals to solicit information regarding the following processes: leadership activities, clinical transformation processes, project management activities, implementation processes, evaluation metrics, terminology and other standards used, and methods used to facilitate end-user adoption. This poster will report the results of this survey. Implications for Nurses will be discussed. The results are valuable to nursing informatics professionals who plan to implement systems and to their nursing executives responsible for the systems that are implemented.

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.