Crystallographic and Computational Insights into Isoform-Selective Dynamics in Nitric Oxide Synthase.

In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors.

Potent, Selective, and Membrane Permeable 2-Amino-4-Substituted Pyridine-Based Neuronal Nitric Oxide Synthase Inhibitors.

A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS), based on a difluorobenzene ring linked to a 2-aminopyridine scaffold with different functionalities at the 4-position, is reported. In our efforts to develop novel nNOS inhibitors for the treatment of neurodegenerative diseases, we discovered 17, which showed excellent potency toward both rat (Ki 15 nM) and human nNOS (Ki 19 nM), with 1075-fold selectivity over human eNOS and 115-fold selectivity over human iNOS. 17 also showed excellent permeability (Pe = 13.7 × 10-6 cm s-1), a low efflux ratio (ER 0.48), along with good metabolic stability in mouse and human liver microsomes, with half-lives of 29 and >60 min, respectively. X-ray cocrystal structures of inhibitors bound with three NOS enzymes, namely, rat nNOS, human nNOS, and human eNOS, revealed detailed structure-activity relationships for the observed potency, selectivity, and permeability properties of the inhibitors.

Essential Paralogous Proteins as Potential Antibiotic Multitargets in Escherichia coli.

Antimicrobial resistance threatens our current standards of care for the treatment and prevention of infectious disease. Antibiotics that have multiple targets have a lower propensity for the development of antibiotic resistance than those that have single targets and therefore represent an important tool in the fight against antimicrobial resistance. In this work, groups of essential paralogous proteins were identified in the important Gram-negative pathogen Escherichia coli that could represent novel targets for multitargeting antibiotics. These groups include targets from a broad range of essential macromolecular and biosynthetic pathways, including cell wall synthesis, membrane biogenesis, transcription, translation, DNA replication, fatty acid biosynthesis, and riboflavin and isoprenoid biosynthesis. Importantly, three groups of clinically validated antibiotic multitargets were identified using this method: the two subunits of the essential topoisomerases, DNA gyrase and topoisomerase IV, and one pair of penicillin-binding proteins. An additional eighteen protein groups represent potentially novel multitargets that could be explored in drug discovery efforts aimed at developing compounds having multiple targets in E. coli and other bacterial pathogens. IMPORTANCE Many types of bacteria have gained resistance to existing antibiotics used in medicine today. Therefore, new antibiotics with novel mechanisms must continue to be developed. One tool to prevent the development of antibiotic resistance is for a single drug to target multiple processes in a bacterium so that more than one change must arise for resistance to develop. The work described here provides a comprehensive search for proteins in the bacterium Escherichia coli that could be targets for such multitargeting antibiotics. Several groups of proteins that are already targets of clinically used antibiotics were identified, indicating that this approach can uncover clinically relevant antibiotic targets. In addition, eighteen currently unexploited groups of proteins were identified, representing new multitargets that could be explored in antibiotic research and development.

2-Aminopyridines with a shortened amino sidechain as potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors.

A series of potent, selective, and highly permeable human neuronal nitric oxide synthase inhibitors (hnNOS) based on the 2-aminopyridine scaffold with a shortened amino sidechain is reported. A rapid and simple protocol was developed to access these inhibitors in excellent yields. Neuronal nitric oxide synthase (nNOS) is a novel therapeutic target for the treatment of various neurological disorders. The major challenges in designing nNOS inhibitors in humans focus on potency, selectivity over other isoforms of nitric oxide synthases (NOSs), and blood-brain barrier permeability. In this context, we discovered a promising inhibitor, 6-(3-(4,4-difluoropiperidin-1-yl)propyl)-4-methylpyridin-2-amine dihydrochloride, that exhibits excellent potency for rat (Ki = 46 nM) and human nNOS (Ki = 48 nM), respectively, with 388-fold human eNOS and 135-fold human iNOS selectivity. It also displayed excellent permeability (Pe = 17.3 × 10-6 cm s-1) through a parallel artificial membrane permeability assay, a model for blood-brain permeability. We found that increasing lipophilicity by incorporation of fluorine atoms on the backbone of the inhibitors significantly increased potential blood-brain barrier permeability. In addition to measuring potency, isoform selectivity, and permeability of NOS inhibitors, we also explored structure-activity relationships via structures of key inhibitors complexed to various isoforms of nitric oxide synthases.

Full Genome Sequence of a Reassortant Human G9P[4] Rotavirus Strain.

This is a report of the complete genomic sequence of a reassortant rotavirus group A G9-P[4]-I2-R2-C2-M2-A2-N2-T2-E6-H2 strain designated RVA/Human-wt/USA/ LB1562/2010/G9P[4].

Prolonged survival in an aged Labrador retriever with a metastatic insulinoma.

This case report highlights an unusually prolonged, asymptomatic, disease-free interval in an aged male Labrador retriever that underwent partial pancreatectomy for a functionally active pancreatic insulinoma with histologically confirmed hepatic metastasis. The patient developed pancreatitis and nonseptic suppurative peritonitis 24 hr after surgical resection of the insulinoma and was managed medically until discharge. Three mo after surgery, the dog was diagnosed with exocrine pancreatic insufficiency (EPI) that was effectively managed with parenteral pancreatic enzymes. Due to normal glucose levels 3 mo postsurgically, liver samples from the initial surgery were resubmitted for immunohistochemistry. Results confirmed insulinoma metastasis with insulin expression. Ten mo postsurgically, the blood glucose was normal and serum insulin levels were slightly above the upper reference limit. The first hypoglycemic episode was documented 23 mo postoperatively, which was effectively managed with prednisone. The cause for the prolonged disease remission and survival was unknown, but was possibly a result of pancreatitis and peritonitis, partial spontaneous regression of metastatic lesions, or idiopathic. Despite life-threatening postoperative complications, this patient enjoyed a profoundly longer than expected survival. This case highlights the importance of removing the primary tumor (insulinoma) despite the presence of metastatic disease.

Laparoscopic vasectomy in African savannah elephant (Loxodonta africana); surgical technique and results.

Several small, enclosed reserves in southern Africa are experiencing significant elephant population growth, which has resulted in associated environmental damage and changes in biodiversity. Although several techniques exist to control elephant populations, e.g., culling, relocation, and immunocontraception, the technique of laparoscopic vasectomy of free-ranging bull elephants was investigated. Bilateral vasectomies were performed in 45 elephants. Of these elephants, one died within 24 hr of recovery and two had complications during surgery but recovered uneventfully. Histologic examination confirmed the resected tissue as ductus deferens in all the bulls. Most animals recovered uneventfully and showed no abnormal behavior after surgery. Complications recorded included incisional dehiscence, 1 full-thickness and 2 partial-thickness lacerations of the large intestine, and initial sling-associated complications, for example, deep radial nerve paresis. One bull was found dead 6 weeks after surgery without showing any prior abnormal signs. Vasectomy in free-ranging African bull elephants may be effectively performed in their normal environment. The surgical procedure can be used as a realistic population management tool in free-ranging elephants without major anesthetic, surgical, or postoperative complications.

Biochemical characterization of DNA-binding proteins from Pyrobaculum aerophilum and Aeropyrum pernix.

Several representatives of the Crenarchaeal branch of the Archaea contain highly abundant, small, positively charged proteins exemplified by the Sso7d protein from Sulfolobus solfataricus. These proteins bind to DNA in a non-sequence-specific manner. Using publicly available genomic sequence information, we identified a second class of small Crenarchaeal DNA-binding proteins represented by the Pyrobaculum aerophilum open reading frame 3192-encoded (Pae3192) protein and its paralogs. We investigated the biochemical properties of the Pae3192 protein and an orthologous protein (Ape1322b) from Aeropyrum pernix in side-by-side experiments with the Sso7d protein. We demonstrate that the recombinant Ape1322b, Pae3192 and Sso7d proteins bind to DNA and that the DNA-protein complexes formed are slightly different for each protein. We show that like Sso7d, Pae3192 constrains negative supercoils in DNA. In addition, we show that all three proteins raise the melting temperature of duplex DNA upon binding. Finally, we present the equilibrium affinity constants and kinetic association constants of each protein for single-stranded and double-stranded DNA.

A genetic selection for supercoiling mutants of Escherichia coli reveals proteins implicated in chromosome structure.

Chromosomes are divided into topologically independent regions, called domains, by the action of uncharacterized barriers. With the goal of identifying domain barrier components, we designed a genetic selection for mutants with reduced negative supercoiling of the Escherichia coli chromosome. We employed a strain that contained two chromosomally located reporter genes under the control of a supercoiling-sensitive promoter and used transposon mutagenesis to generate a wide range of mutants. We subjected the selected mutants to a series of secondary screens and identified five proteins as modulators of chromosomal supercoiling in vivo. Three of these proteins: H-NS, Fis and DksA, have clear ties to chromosome biology. The other two proteins, phosphoglucomutase (Pgm) and transketolase (TktA), are enzymes involved in carbohydrate metabolism and have not previously been shown to affect DNA. Deletion of any of the identified genes specifically affected chromosome topology, without affecting plasmid supercoiling. We suggest that at least H-NS, Fis and perhaps TktA assist directly in the supercoiling of domains by forming topological barriers on the E. coli chromosome.

Topological domain structure of the Escherichia coli chromosome.

The circular chromosome of Escherichia coli is organized into independently supercoiled loops, or topological domains. We investigated the organization and size of these domains in vivo and in vitro. Using the expression of >300 supercoiling-sensitive genes to gauge local chromosomal supercoiling, we quantitatively measured the spread of relaxation from double-strand breaks generated in vivo and thereby calculated the distance to the nearest domain boundary. In a complementary approach, we gently isolated chromosomes and examined the lengths of individual supercoiled loops by electron microscopy. The results from these two very different methods agree remarkably well. By comparing our results to Monte Carlo simulations of domain organization models, we conclude that domain barriers are not placed stably at fixed sites on the chromosome but instead are effectively randomly distributed. We find that domains are much smaller than previously reported, approximately 10 kb on average. We discuss the implications of these findings and present models for how domain barriers may be generated and displaced during the cell cycle in a stochastic fashion.

Disentangling DNA during replication: a tale of two strands.

The seminal papers by Watson and Crick in 1953 on the structure and function of DNA clearly enunciated the challenge their model presented of how the intertwined strands of DNA are unwound and separated for replication to occur. We first give a historical overview of the major discoveries in the past 50 years that address this challenge. We then describe in more detail the cellular mechanisms responsible for the unlinking of DNA. No single strategy on its own accounts for the complete unlinking of chromosomes required for DNA segregation to proceed. Rather, it is the combined effects of topoisomerase action, chromosome organization and DNA-condensing proteins that allow the successful partitioning of chromosomes into dividing cells. Finally, we propose a model of chromosome structure, consistent with recent findings, that explains how the problem of unlinking is alleviated by the division of chromosomal DNA into manageably sized domains.

Agreement between patient self-report and a Veterans Affairs national pharmacy database for identifying recent exposures to antibiotics.

PURPOSE: The dramatic rise in antibiotic drug resistance among community pathogens has stimulated interest in the epidemiological relationship between antibiotic exposure and drug resistance. In assessing the strength of this relationship, studies are hampered by the lack of data on the accuracy of subject self-report of antibiotic exposure. The authors compared self-report with pharmacy dispensing data to determine the accuracy of self-reported antibiotic exposure. METHODS: The study design was a cross-sectional survey of veterans seen at the Philadelphia Veterans Affairs (VA) Medical Center in 1999 and 2000. Subjects reported exposures to antibiotics, antihypertensive drugs and nonsteroidal anti-inflammatory drugs through a structured telephone interview. The instrument included open-ended questions, condition-specific prompts and drug-specific prompts. Subject responses were linked to a national VA pharmacy database that served as the reference standard for evaluating self-reported exposures. RESULTS: The authors found that the sensitivity of self-report of antibiotic exposure increased with increasing use of prompts. A comprehensive assessment of antibiotic exposure identified 73% of antibiotic exposures, compared to 73% of antihypertensive drug exposures and 92% of nonsteroidal anti-inflammatory drug exposures. CONCLUSIONS: Assessment of antibiotic exposure appears to be comparable to assessment of other chronic and episodic drugs. Multistep assessment of exposure improves the sensitivity of assessment.

Alteration of Escherichia coli topoisomerase IV to novobiocin resistance.

DNA gyrase and topoisomerase IV (topo IV) are the two essential type II topoisomerases of Escherichia coli. Gyrase is responsible for maintaining negative supercoiling of the bacterial chromosome, whereas topo IV's primary role is in disentangling daughter chromosomes following DNA replication. Coumarins, such as novobiocin, are wide-spectrum antimicrobial agents that primarily interfere with DNA gyrase. In this work we designed an alteration in the ParE subunit of topo IV at a site homologous to that which confers coumarin resistance in gyrase. This parE mutation renders the encoded topo IV approximately 40-fold resistant to inhibition by novobiocin in vitro and imparts a similar resistance to inhibition of topo IV-mediated relaxation of supercoiled DNA in vivo. We conclude that topo IV is a secondary target of novobiocin and that it is very likely to be inhibited by the same mechanism as DNA gyrase.

Use of EBV PCR for the diagnosis and monitoring of post-transplant lymphoproliferative disorder in adult solid organ transplant patients.

Epstein-Barr virus (EBV) is known to be involved in the majority of patients who develop post-transplant lymphoproliferative disorder after solid organ transplant. We conducted a retrospective study to determine the utility of qualitative and quantitative Epstein-Barr virus polymerase chain reaction (PCR) for the diagnosis and monitoring of post-transplant lymphoproliferative disorder in adult solid organ transplant patients. Peripheral blood leukocytes obtained from 35 adult solid organ transplant patients consecutively referred for evaluation of possible post-transplant lymphoproliferative disorder, were tested by EBV PCR at the time of initial evaluation and at time points thereafter. Eighteen of 35 (51%) patients were ultimately diagnosed with post-transplant lymphoproliferative disorder by tissue biopsy. Fifteen of 18 (83%) patients were found to have EBER-1 positive tumors by in situ hybridization. EBV PCR was positive in 7 of 15 patients, suggesting a sensitivity of 39%. Seventeen patients without post-transplant lymphoproliferative disorder and three with EBER-1 negative post-transplant lymphoproliferative disorder all had negative EBV PCR tests, suggesting a specificity of 100%. We observed that declines in EBV DNA load were associated with response to therapeutic interventions, such as reduction in immunosuppression, rituximab therapy and chemotherapy. We conclude that peripheral blood EBV PCR may have a role in the diagnosis and monitoring of post-transplant lymphoproliferative disorder in adult solid organ transplant patients.

Effect of blood collection technique in mice on clinical pathology parameters.

A study was conducted in normal healthy C57BL/6 mice to determine the effect of method of blood collection on clinical pathology parameters and to provide value ranges for these parameters. Males and females were used and were randomly assigned to treatment groups based upon phlebotomy method. The blood was collected using one of four methods: intracardiac (IC), a single attempt at collection from the caudal vena cava (VC), collection from the caudal vena cava with collection of any extravasated blood from the peritoneum (MC), or retroorbital phlebotomy (RO). Evaluation of blood and serum samples was conducted for a number of serum biochemistries including liver function tests and complete blood count with differentials and platelet counts. Female mice demonstrated higher values for red blood cell number, hemoglobin (p < 0.002), hematocrit, alkaline phosphatase, albumin, total protein, and creatinine. Males demonstrated higher values for platelet counts, specific white blood cell numbers (total, neutrophil, lymphocyte, and eosinophil counts), globulin, amylase, and the BUN/creatinine ratio. Overall, the VC method was associated with the least variation in both sexes and appeared slightly better than the IC method for the parameters evaluated. The largest difference between groups was noted for the transaminase levels. While alanine aminotransferase (ALT) values were similar between the IC and VC groups, aspartate aminotransferase (AST) values were associated with less variation for the VC method. Transaminase levels for the MC and RO groups were associated with relatively large ranges and variation. ALT results from the RO method, the only method amenable to repetitive sample collection used in this evaluation, indicate that this is an acceptable method. The results demonstrate the substantial impact that phlebotomy method has on the assay results and that the VC or IC methods provide the most consistent results. The ranges by collection method and sex provided here can be used to select the preferred method of collection when designing a study and for comparison of data obtained with reference ranges. The authors recommend establishment of normal ranges based upon methods employed within an investigator's laboratory.

Pitfalls in predicting resting energy requirements in critically ill children: a comparison of predictive methods to indirect calorimetry.

BACKGROUND: Critical illness in children is thought to have profound effects on nutritional status. It is essential to avoid complications associated with inadequate nutrition support and delivery of excess energy. OBJECTIVE: To compare the results of several commonly used methods for predicting energy requirements in a group of critically ill children indirect calorimetry was used to measure energy expenditure in these children. DESIGN: Resting energy expenditures estimated by different prediction methods for energy were compared with measurements of actual resting energy expenditure obtained by indirect calorimetry in 52 children admitted to a pediatric intensive care unit. Agreement between each predictive method and indirect calorimetry was evaluated by Bland-Altman limits of agreement and by whether the methods met the predetermined criterion for accuracy of within 10% of the measured value. RESULTS: None of the equations predicted individual values accurately. Each of the predictive equations gave a wide and variable scatter of predicted values around the median. The recommended dietary allowance for energy was the least accurate and differed significantly even from the other predictive methods, overestimating energy expenditure in 50 of 52 patients. None of the remaining methods stood out as being more precise. CONCLUSIONS: Predictive methods commonly used to estimate energy expenditure in critically ill children are very imprecise and may lead to overprovision or underprovision of nutrition support. Resting energy expenditure should be measured by indirect calorimetry whenever possible.