Designer broad-spectrum polyimidazolium antibiotics

For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive Staphylococcus aureus we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a site-directed menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen Pseudomonas aeruginosa, PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections.

AMPs and AMP mimics have attracted considerable attention as candidates for therapeutic development (1). The basic design elements include a region of charged residues, generally cationic residues, enabling interaction with bacterial cell surfaces, combined with a hydrophobic nature in AMPs (2). Unfortunately, AMPs and related polymers, in general, have one or more issues that limit their use as broad-spectrum antibiotics. Some are quite toxic to human cells, the potency of some is not adequate for human administration, others are sensitive to salt at levels present in human fluids, and some are too difficult and expensive to synthesize (3, 4). One broad-spectrum antimicrobial peptide, CST has seen increased recent use as a last resort antibiotic. CST is believed to kill bacteria by virtue of its ability to disrupt membrane integrity (5). This antibiotic requires intravenous administration and is nephrotoxic (6). The emergence of CST-resistant pathogens has also become a significant problem (7). We are unaware of any new broad-spectrum AMPs that have advanced to clinical trials.Imidazolium (IM) salts are antimicrobials (8), and there is an emerging literature on antimicrobial activity of side-chain and main-chain polyimidazolium (PIM) salts with chemical structures that are in some ways similar to those we describe. Although PIMs are potent antimicrobials, there are biocompatibility problems hindering their development, and some have somewhat limited activity spectra. As with other AMPs, there have been toxicity issues, potency issues, and delivery issues as many have large molecular masses, and there is little known about mammalian cell toxicity or mechanism of action (9–12).Here we show that members of a series of PIMs we designed and synthesized are potent broad-spectrum antibacterial compounds. We selected two for further analysis and showed they retain activity even against pan-antibiotic-resistant bacteria. Unlike CST and many other AMPs, which disrupt bacterial membranes, our model PIM is bactericidal without disrupting bacterial membranes. Our experiments provide insights about mechanism of action, the potential for the emergence of PIM resistance, and indicate PIMs are effective against a model gram-negative and a model gram-positive pathogen in murine infection models.     

Low density lipoprotein receptor activity in freshly isolated human blood monocytes and lymphocytes

Circulating human monocytes and lymphocytes were isolated by counterflow and density gradient centrifugation. Binding and degradation of low density lipoprotein (LDL) occurred predominantly in monocytes and to a much lesser extent in lymphocytes. The findings are consistent with greater LDL receptor activity in freshly isolated monocytes than lymphocytes, in keeping with differences in other cell surface receptors between these two cell types. Therefore, when freshly isolated mixed mononuclear cells are used to study LDL receptor activity in vivo in humans, careful attention needs to be given to the proportions of monocytes and lymphocytes, or alternatively, relatively pure preparations of monocytes should be used.     

Effect of adrenocorticotropin administration on the biosynthesis of corticosteroid-binding globulin in fetal sheep

Parturition in sheep is initiated by the fetus through activation of the fetal hypothalamic-pituitary-adrenal axis and is associated with increased concentrations of ACTH, cortisol, and corticosteroid-binding globulin (CBG) in the fetal circulation during the final 10-15 days of pregnancy. Premature parturition and a precocious elevation in fetal plasma CBG are produced by intrafetal ACTH administration, but the possible sources of CBG in the ovine fetus are not known. To determine these sites, CBG mRNA was measured in tissues from fetal sheep in late pregnancy and after intrafetal ACTH treatment, using a sheep CBG cDNA. Fetal ACTH treatment caused a significant increase in the fetal plasma corticosteroid-binding capacity (CBC), although there was no significant difference in CBC between umbilical arterial and umbilical venous plasma. After ACTH treatment, CBC was elevated in fetal liver and kidney. Cortisol binding in these tissues had characteristics similar to those of cortisol binding in fetal sheep plasma. By Northern blot analysis a single mRNA (1.7 kilobases) for CBG was detected in fetal liver, kidney, lung, and adrenal, but not in placenta. The abundance of CBG mRNA in the fetal liver was greater than that in other tissues, but was unchanged by ACTH treatment. The level of CBG mRNA in the fetal kidney, but not in other tissues, increased 3-fold after ACTH. We conclude that the elevation in plasma CBC after intrafetal ACTH, and presumably also at term pregnancy, does not reflect production of CBC by the placenta or transfer from the mother. Rather, it results from production primarily in the fetal liver and kidney, although only in the latter tissue is CBG mRNA accumulation increased by intrafetal ACTH treatment.     

Expression of factor VIII in recombinant and transgenic systems

Deficiency in a coagulation factor VIII (FVIII) causes a genetic disorder hemophilia A, which is treated by repeated infusions of expensive FVIII products. Recombinant FVIII (rFVIII), the culmination of years of extensive international research, is an important alternative to plasma-derived FVIII (pdFVIII) and is considered to have a higher margin of safety. Advances in biotechnology allowed production of rFVIII at industrial scale, which significantly improved treatment of hemophilia A patients. We review the contemporary methods used for FVIII expression in mammalian cell culture systems and discuss the factors responsible for insufficient recoveries of rFVIII, such as inefficient accumulation of FVIII mRNA in the cell, complexity of the mechanisms of FVIII secretion, and instability of secreted FVIII. The approaches to improve the yield of rFVIII in cell culture systems include genetic engineering of B-domain-deleted FVIII, introduction of introns into FVIII cDNA constructs for more efficient processing and accumulation of FVIII mRNA, and introduction of mutations into chaperone-binding sites of FVIII to improve its secretion. Design of FVIII with prolonged half-life in vivo is considered as another promising direction in improving rFVIII protein and efficiency of hemophilia A therapy. As an alternative to expression of rFVIII in cell culture systems, we discuss production of rFVIII in transgenic animals, where high levels of rFVIII have been successfully secreted into milk. We also pay attention to the major limitations of this approach, such as safety issues associated with potential transmission of animal pathogens. Finally, we present a brief characterization of commercial recombinant FVIII products currently available on the market for hemophilia A treatment.     

A new configuration for right ventricular assist with skeletal muscle ventricle. Short-term studies

BACKGROUND. Previous attempts to provide right heart assistance with skeletal muscle ventricles (SMVs) have been frustrated by the low preload supplied by the systemic venous blood pressure. In the present study, right ventricular pressure was exploited to provide more optimal preload, the SMV being connected by valved conduits between right ventricular free wall and the main pulmonary artery. METHODS AND RESULTS. SMVs were constructed from the right latissimus dorsi muscle in seven mongrel dogs. Following a delay period of 4 weeks, SMVs were preconditioned with 2-Hz continuous stimulation for 5-6 weeks. The SMV was then connected to the right ventricle using a porcine valved Dacron conduit. A similar valved conduit connected the SMV to the main pulmonary artery that had been ligated proximally. SMVs were stimulated with 33-Hz burst frequency to contract synchronously with ventricular diastole in a 1:2 mode. The stimulator was intermittently turned off to permit comparison of assisted and nonassisted circulation. Cardiac output increased by 27% at 1 hour (1,437 +/- 54 versus 1,140 +/- 64 ml/min, p less than 0.005) and by 30% at 4 hours (1,403 +/- 161 versus 1,074 +/- 99 ml/min, p less than 0.005), systemic arterial systolic pressure increased at 1 hour by 12% (87.1 +/- 4.9 versus 78.0 +/- 4.9 mm Hg, p less than 0.05) and by 13% at 4 hours (81.4 +/- 2.8 versus 72.3 +/- 3.4 mm Hg, p less than 0.005), and peak pulmonary arterial pressure increased at 1 hour by 35% (28.0 +/- 2.1 versus 20.9 +/- 1.8 mm Hg, p less than 0.01) and by 37% at 4 hours (31.5 +/- 2.6 versus 23.0 +/- 0.4 mm Hg, p less than 0.05). Peak SMV pressure was 52.8 +/- 2.0 mm Hg at 1 hour and 49.9 +/- 3.3 mm Hg at 4 hours (p = NS). CONCLUSIONS. The improved preload supplied by this configuration of right ventricular assist enabled an SMV to provide stable and effective circulatory support throughout the 4-hour duration of the experiment.     

Innovative information visualization of electronic health record data: a systematic review

Objective This study investigates the use of visualization techniques reported between 1996 and 2013 and evaluates innovative approaches to information visualization of electronic health record (EHR) data for knowledge discovery.Methods An electronic literature search was conducted May–July 2013 using MEDLINE and Web of Knowledge, supplemented by citation searching, gray literature searching, and reference list reviews. General search terms were used to assure a comprehensive document search.Results Beginning with 891 articles, the number of articles was reduced by eliminating 191 duplicates. A matrix was developed for categorizing all abstracts and to assist with determining those to be excluded for review. Eighteen articles were included in the final analysis.Discussion Several visualization techniques have been extensively researched. The most mature system is LifeLines and its applications as LifeLines2, EventFlow, and LifeFlow. Initially, research focused on records from a single patient and visualization of the complex data related to one patient. Since 2010, the techniques under investigation are for use with large numbers of patient records and events. Most are linear and allow interaction through scaling and zooming to resize. Color, density, and filter techniques are commonly used for visualization.Conclusions With the burgeoning increase in the amount of electronic healthcare data, the potential for knowledge discovery is significant if data are managed in innovative and effective ways. We identify challenges discovered by previous EHR visualization research, which will help researchers who seek to design and improve visualization techniques.     

The psychometric properties of the Evaluation of Daily Activity Questionnaire in seven musculoskeletal conditions

Purpose: The purpose of this study is to psychometrically test the Evaluation of Daily Activity Questionnaire in seven musculoskeletal conditions.

Materials and methods: One thousand and two hundred people with ankylosing spondylitis; osteoarthritis; systemic lupus erythematosus; systemic sclerosis; chronic pain; chronic upper limb disorders; or Primary Sjögren’s syndrome completed the Evaluation of Daily Activity Questionnaire, Health Assessment Questionnaire and Short-Form Health Survey v2. We examined internal construct validity using Rasch analysis, internal consistency, concurrent validity with the Health Assessment Questionnaire and Short-Form Health Survey v2. Participants repeated the Evaluation of Daily Activity Questionnaire to assess test–retest reliability.

Results: The 12 domains satisfied Rasch model expectations for fit, local dependency, unidimensionality and invariance by age and gender, in each musculoskeletal condition. Internal consistency was consistent with individual use (Cronbach’s α?>?0.90); concurrent validity was strong (Health Assessment Questionnaire:?r_s?=?0.60–0.92; Short-Form Health Survey v2 Physical Function:?r_s?=??0.61 to ?0.91) and test–retest reliability excellent (Intra-Class Correlation Coefficient(2,1)?=?0.77–0.96).

Conclusion: The Evaluation of Daily Activity Questionnaire satisfied Rasch model requirements for construct validity and has good reliability and validity in each MSC. The Evaluation of Daily Activity Questionnaire can be used as a measure of everyday activity in practice and research with people with musculoskeletal conditions.

• Implications for rehabilitation

• The Evaluation of Daily Activity Questionnaire evaluates users’ ability to perform common daily activities (in 12 domains) that were identified as problematic by people with seven musculoskeletal conditions (i.e., osteoarthritis, systemic lupus, ankylosing spondylitis, chronic pain, chronic upper limb conditions, systemic sclerosis, Sjogren’s syndrome).

• Most patients considered the Evaluation of Daily Activity Questionnaire was the right length and would be helpful for discussing everyday problems with an occupational therapist.

• The 12 domains have good reliability and validity and can be combined into two components: Self-Care and Mobility.

• The Evaluation of Daily Activity Questionnaire is suitable for use both in clinical practice and research and a User Manual is available for therapists and researchers.