Antimicrobial Peptides and Copper(II) Ions: Novel Therapeutic Opportunities.

The emergence of new pathogens and multidrug resistant bacteria is an important public health issue that requires the development of novel classes of antibiotics. Antimicrobial peptides (AMPs) are a promising platform with great potential for the identification of new lead compounds that can combat the aforementioned pathogens due to their broad-spectrum antimicrobial activity and relatively low rate of resistance emergence. AMPs of multicellular organisms made their debut four decades ago thanks to ingenious researchers who asked simple questions about the resistance to bacterial infections of insects. Questions such as "Do fruit flies ever get sick?", combined with pioneering studies, have led to an understanding of AMPs as universal weapons of the immune system. This review focuses on a subclass of AMPs that feature a metal binding motif known as the amino terminal copper and nickel (ATCUN) motif. One of the metal-based strategies of hosts facing a pathogen, it includes wielding the inherent toxicity of copper and deliberately trafficking this metal ion into sites of infection. The sudden increase in the concentration of copper ions in the presence of ATCUN-containing AMPs (ATCUN-AMPs) likely results in a synergistic interaction. Herein, we examine common structural features in ATCUN-AMPs that exist across species, and we highlight unique features that deserve additional attention. We also present the current state of knowledge about the molecular mechanisms behind their antimicrobial activity and the methods available to study this promising class of AMPs.

Roles of Virtual Screening and Molecular Dynamics Simulations in Discovering and Understanding Antimalarial Drugs.

Malaria continues to be a global health threat, with approximately 247 million cases worldwide. Despite therapeutic interventions being available, patient compliance is a problem due to the length of treatment. Moreover, drug-resistant strains have emerged over the years, necessitating urgent identification of novel and more potent treatments. Given that traditional drug discovery often requires a great deal of time and resources, most drug discovery efforts now use computational methods. In silico techniques such as quantitative structure-activity relationship (QSAR), docking, and molecular dynamics (MD) can be used to study protein-ligand interactions and determine the potency and safety profile of a set of candidate compounds to help prioritize those tested using assays and animal models. This paper provides an overview of antimalarial drug discovery and the application of computational methods in identifying candidate inhibitors and elucidating their potential mechanisms of action. We conclude with the continued challenges and future perspectives in the field of antimalarial drug discovery.

The DNA sequence and biological annotation of human chromosome 1.

The reference sequence for each human chromosome provides the framework for understanding genome function, variation and evolution. Here we report the finished sequence and biological annotation of human chromosome 1. Chromosome 1 is gene-dense, with 3,141 genes and 991 pseudogenes, and many coding sequences overlap. Rearrangements and mutations of chromosome 1 are prevalent in cancer and many other diseases. Patterns of sequence variation reveal signals of recent selection in specific genes that may contribute to human fitness, and also in regions where no function is evident. Fine-scale recombination occurs in hotspots of varying intensity along the sequence, and is enriched near genes. These and other studies of human biology and disease encoded within chromosome 1 are made possible with the highly accurate annotated sequence, as part of the completed set of chromosome sequences that comprise the reference human genome.

DNA sequence and analysis of human chromosome 9.

Chromosome 9 is highly structurally polymorphic. It contains the largest autosomal block of heterochromatin, which is heteromorphic in 6-8% of humans, whereas pericentric inversions occur in more than 1% of the population. The finished euchromatic sequence of chromosome 9 comprises 109,044,351 base pairs and represents >99.6% of the region. Analysis of the sequence reveals many intra- and interchromosomal duplications, including segmental duplications adjacent to both the centromere and the large heterochromatic block. We have annotated 1,149 genes, including genes implicated in male-to-female sex reversal, cancer and neurodegenerative disease, and 426 pseudogenes. The chromosome contains the largest interferon gene cluster in the human genome. There is also a region of exceptionally high gene and G + C content including genes paralogous to those in the major histocompatibility complex. We have also detected recently duplicated genes that exhibit different rates of sequence divergence, presumably reflecting natural selection.

The DNA sequence and comparative analysis of human chromosome 10.

The finished sequence of human chromosome 10 comprises a total of 131,666,441 base pairs. It represents 99.4% of the euchromatic DNA and includes one megabase of heterochromatic sequence within the pericentromeric region of the short and long arm of the chromosome. Sequence annotation revealed 1,357 genes, of which 816 are protein coding, and 430 are pseudogenes. We observed widespread occurrence of overlapping coding genes (either strand) and identified 67 antisense transcripts. Our analysis suggests that both inter- and intrachromosomal segmental duplications have impacted on the gene count on chromosome 10. Multispecies comparative analysis indicated that we can readily annotate the protein-coding genes with current resources. We estimate that over 95% of all coding exons were identified in this study. Assessment of single base changes between the human chromosome 10 and chimpanzee sequence revealed nonsense mutations in only 21 coding genes with respect to the human sequence.

Antimicrobial Susceptibility Testing of Antimicrobial Peptides to Better Predict Efficacy.

During the development of antimicrobial peptides (AMP) as potential therapeutics, antimicrobial susceptibility testing (AST) stands as an essential part of the process in identification and optimisation of candidate AMP. Standard methods for AST, developed almost 60 years ago for testing conventional antibiotics, are not necessarily fit for purpose when it comes to determining the susceptibility of microorganisms to AMP. Without careful consideration of the parameters comprising AST there is a risk of failing to identify novel antimicrobials at a time when antimicrobial resistance (AMR) is leading the planet toward a post-antibiotic era. More physiologically/clinically relevant AST will allow better determination of the preclinical activity of drug candidates and allow the identification of lead compounds. An important consideration is the efficacy of AMP in biological matrices replicating sites of infection, e.g., blood/plasma/serum, lung bronchiolar lavage fluid/sputum, urine, biofilms, etc., as this will likely be more predictive of clinical efficacy. Additionally, specific AST for different target microorganisms may help to better predict efficacy of AMP in specific infections. In this manuscript, we describe what we believe are the key considerations for AST of AMP and hope that this information can better guide the preclinical development of AMP toward becoming a new generation of urgently needed antimicrobials.

A Potent Host Defense Peptide Triggers DNA Damage and Is Active against Multidrug-Resistant Gram-Negative Pathogens.

Gram-negative bacteria are some of the biggest threats to public health due to a large prevalence of antibiotic resistance. The difficulty in treating bacterial infections, stemming from their double membrane structure combined with efflux pumps in the outer membrane, has resulted in a much greater need for antimicrobials with activity against these pathogens. Tunicate host defense peptide (HDP), Clavanin A, is capable of not only inhibiting Gram-negative growth but also potentiating activity in the presence of Zn(II). Here, we provide evidence that the improvements of Clavanin A activity in the presence of Zn(II) are due to its novel mechanism of action. We employed E. coli TD172 (ΔrecA::kan) and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to show in cellulae that DNA damage occurs upon treatment with Clavanin A. In vitro assays demonstrated that Zn(II) ions are required for the nuclease activity of the peptide. The quantum mechanics/molecular mechanics (QM/MM) calculations were used to investigate the mechanism of DNA damage. In the rate-determining step of the proposed mechanism, due to its Lewis acidity, the Zn(II) ion activates the scissile P-O bond of DNA and creates a hydroxyl nucleophile from a water molecule. A subsequent attack by this group to the electrophilic phosphorus cleaves the scissile phosphoester bond. Additionally, we utilized bacterial cytological profiling (BCP), circular dichroism (CD) spectroscopy in the presence of lipid vesicles, and surface plasmon resonance combined with electrical impedance spectroscopy in order to address the apparent discrepancies between our results and the previous studies regarding the mechanism of action of Clavanin A. Finally, our approach may lead to the identification of additional Clavanin A like HDPs and promote the development of antimicrobial peptide based therapeutics.

Effect of smoking on the accumulation of deficits, frailty and survival in older adults: a secondary analysis from the Canadian Study of Health and Aging.

OBJECTIVES: Smoking has adverse effects on a variety of organ systems but little is known about the relationship between smoking and frailty. We aimed to investigate differences in health status between smoking and non smoking older adults. DESIGN AND SETTING: The Canadian Study of Health and Aging, a nationally representative cohort study. PARTICIPANTS: Nine thousand and eight community-dwelling men and women age 65 years and over at baseline. MEASUREMENTS: Smoking status was determined using a Self-Assessed Risk Factor Questionnaire. Comparisons were made between never smokers, light smokers and heavy smokers with heavy smokers defined as those who smoked >or= 1 pack per day for 20 years or more. A frailty index (FI) generated from 40 self-reported health deficits was also modified to exclude 5 variables that could be directly attributed to smoking (e.g. cough). Decedent information was collected over 10 years. RESULTS: Average FI values increased exponentially with age. For both men and women, heavy smokers were the most frail, light smokers had intermediate frailty status and never smokers were fittest. Modification of the FI did not impact these differences. Heavy smokers had significantly worse mortality than non smokers and higher rates of death in smokers persisted in the oldest old. 120 month survival curves, grouped for age, sex and smoking status showed that male smokers > 75 years had the highest mortality rates. CONCLUSIONS: Smoking causes poorer health status at older ages which can be captured by the frailty index. Higher rates of death in smokers persist in the oldest old, with no emergence of "survivors" with fitness or longevity advantages.

Molecular Dynamics Investigation into the Effect of Zinc(II) on the Structure and Membrane Interactions of the Antimicrobial Peptide Clavanin A.

Clavanin A (ClavA) is an antimicrobial peptide (AMP) whose antimicrobial activity is enhanced in the presence of Zn(II) ions. The antimicrobial activity of ClavA has been shown to increase 16-fold in the presence of Zn(II) ions. In this study, we investigate the potential sources of this enhancement, namely, the effect of Zn(II) binding on the helical conformation of ClavA and on the ClavA interaction with a model for gram-negative bacterial membranes. In addition, we investigate the effect of Zn(II) on the membrane mechanical properties. We employed all-atom equilibrium molecular dynamics simulations initiated from both fully helical and random coil structures of ClavA. We observe that Zn(II) can stabilize an existing helical conformation in the Zn(II)-binding region, but we do not observe induction of helical conformations in systems initiated in random coil configurations. Zn(II) binding to ClavA provides more favorable electrostatics for membrane association in the C-terminal region. This is evidenced by longer and stronger C-terminal-lipid interactions. Zn(II) is also capable of modulating the membrane properties in a manner which favors ClavA insertion and the potential for enhanced translocation into the cell. This work provides insights into the role of divalent metal cations in the antimicrobial activity of ClavA. This information can be used for the development of synthetic AMPs containing motifs that can bind metals (metalloAMPs) for therapeutic and medical purposes.

Functional and structural basis of E. coli enolase inhibition by SF2312: a mimic of the carbanion intermediate.

Many years ago, the natural secondary metabolite SF2312, produced by the actinomycete Micromonospora, was reported to display broad spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria. Recent studies have revealed that SF2312, a natural phosphonic acid, functions as a potent inhibitor of human enolase. The mechanism of SF2312 inhibition of bacterial enolase and its role in bacterial growth and reproduction, however, have remained elusive. In this work, we detail a structural analysis of E. coli enolase bound to both SF2312 and its oxidized imide-form. Our studies support a model in which SF2312 acts as an analog of a high energy intermediate formed during the catalytic process. Biochemical, biophysical, computational and kinetic characterization of these compounds confirm that altering features characteristic of a putative carbanion (enolate) intermediate significantly reduces the potency of enzyme inhibition. When SF2312 is combined with fosfomycin in the presence of glucose-6 phosphate, significant synergy is observed. This suggests the two agents could be used as a potent combination, targeting distinct cellular mechanism for the treatment of bacterial infections. Together, our studies rationalize the structure-activity relationships for these phosphonates and validate enolase as a promising target for antibiotic discovery.

Ensembl 2006.

The Ensembl (http://www.ensembl.org/) project provides a comprehensive and integrated source of annotation of large genome sequences. Over the last year the number of genomes available from the Ensembl site has increased from 4 to 19, with the addition of the mammalian genomes of Rhesus macaque and Opossum, the chordate genome of Ciona intestinalis and the import and integration of the yeast genome. The year has also seen extensive improvements to both data analysis and presentation, with the introduction of a redesigned website, the addition of RNA gene and regulatory annotation and substantial improvements to the integration of human genome variation data.

Perturbed hemopoiesis and the generation of multipotential stem cell clones in src-infected bone marrow cultures is an indirect or transient effect of the oncogene.

Multipotential stem cell lines, derived specifically from long-term bone marrow cultures infected with a recombinant retrovirus carrying v-src, lack v-src. Stable consequences thus result from transient actions or indirect effects of v-src on other cells, with the latter possibility being favored by its mosaic expression in marrow cultures.

The Vertebrate Genome Annotation (Vega) database.

The Vertebrate Genome Annotation (Vega) database (http://vega.sanger.ac.uk) has been designed to be a community resource for browsing manual annotation of finished sequences from a variety of vertebrate genomes. Its core database is based on an Ensembl-style schema, extended to incorporate curation-specific metadata. In collaboration with the genome sequencing centres, Vega attempts to present consistent high-quality annotation of the published human chromosome sequences. In addition, it is also possible to view various finished regions from other vertebrates, including mouse and zebrafish. Vega displays only manually annotated gene structures built using transcriptional evidence, which can be examined in the browser. Attempts have been made to standardize the annotation procedure across each vertebrate genome, which should aid comparative analysis of orthologues across the different finished regions.

Ensembl 2005.

The Ensembl (http://www.ensembl.org/) project provides a comprehensive and integrated source of annotation of large genome sequences. Over the last year the number of genomes available from the Ensembl site has increased by 7 to 16, with the addition of the six vertebrate genomes of chimpanzee, dog, cow, chicken, tetraodon and frog and the insect genome of honeybee. The majority have been annotated automatically using the Ensembl gene build system, showing its flexibility to reliably annotate a wide variety of genomes. With the increased number of vertebrate genomes, the comparative analysis provided to users has been greatly improved, with new website interfaces allowing annotation of different genomes to be directly compared. The Ensembl software system is being increasingly widely reused in different projects showing the benefits of a completely open approach to software development and distribution.

Single Chain Polymeric Nanoparticles to Promote Selective Hydroxylation Reactions of Phenol Catalyzed by Copper.

Metal-containing single chain polymeric nanoparticles (SCPNs) can be used as synthetic mimics of metalloenzymes. Currently, the role of the folded polymer backbones on the activity and selectivity of metal sites is not clear. Herein, we report our findings on how polymeric frameworks modulate the coordination of Cu sites and the catalytic activity/selectivity of Cu-containing SCPNs mimicking monophenol hydroxylation reactions. Imidazole-functionalized copolymers of poly(methyl methacrylate-co-3-imidazolyl-2-hydroxy propyl methacrylate) were used for intramolecular Cu-imidazole binding that triggered the self-folding of polymers. Polymer chains imposed steric hindrance which yielded unsaturated Cu sites with an average coordination number of 3.3. Cu-containing SCPNs showed a high selectivity for the hydroxylation reaction of phenol to catechol, >80%, with a turnover frequency of >870 h-1 at 60 °C. The selectivity was largely influenced by the flexibility of the folded polymer backbone where a more flexible polymer backbone allows the cooperative catalysis of two Cu sites. The second coordination sphere provided by the folded polymer that has been less studied is therefore critical in the design of active mimics of metalloenzymes.

The Ensembl genome database project.

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of the human genome sequence, with confirmed gene predictions that have been integrated with external data sources, and is available as either an interactive web site or as flat files. It is also an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements from sequence analysis to data storage and visualisation. The Ensembl site is one of the leading sources of human genome sequence annotation and provided much of the analysis for publication by the international human genome project of the draft genome. The Ensembl system is being installed around the world in both companies and academic sites on machines ranging from supercomputers to laptops.

Ensembl 2002: accommodating comparative genomics.

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organise biology around the sequences of large genomes. It is a comprehensive source of stable automatic annotation of human, mouse and other genome sequences, available as either an interactive web site or as flat files. Ensembl also integrates manually annotated gene structures from external sources where available. As well as being one of the leading sources of genome annotation, Ensembl is an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements. These range from sequence analysis to data storage and visualisation and installations exist around the world in both companies and at academic sites. With both human and mouse genome sequences available and more vertebrate sequences to follow, many of the recent developments in Ensembl have focusing on developing automatic comparative genome analysis and visualisation.

Ensembl 2004.

The Ensembl (http://www.ensembl.org/) database project provides a bioinformatics framework to organize biology around the sequences of large genomes. It is a comprehensive and integrated source of annotation of large genome sequences, available via interactive website, web services or flat files. As well as being one of the leading sources of genome annotation, Ensembl is an open source software engineering project to develop a portable system able to handle very large genomes and associated requirements. The facilities of the system range from sequence analysis to data storage and visualization and installations exist around the world both in companies and at academic sites. With a total of nine genome sequences available from Ensembl and more genomes to follow, recent developments have focused mainly on closer integration between genomes and external data.

Transcription of Rous sarcoma proviruses in rat cells is determined by chromosomal position effects that fluctuate and can operate over long distances.

Most Rous sarcoma proviruses integrated in rat DNA are inactive. The active minority are not necessarily a consequence of long terminal repeat mutations and their transcription can fluctuate in parallel with previously demonstrated transitions in chromatin configuration. Transcriptional alternations cannot be attributed to variations in positive or negative factors that operate independently of the site of provirus integration. Moreover, distinct proviruses in the same cell can be differentially expressed and flanking cellular elements can act over several kilobases to inhibit provirus transcription. We postulate that position-dependent fluctuations in proviral repression can be mediated by trans-acting factors, and DNA transfections indicate that initiation and maintenance of this phenomenon are separable entities. Provirus activity presumably reflects otherwise inapparent cell controls that affect chromatin structure over long distances.

Comparison of surface accessible residues in human and murine immunoglobulin Fv domains. Implication for humanization of murine antibodies.

Statistical analysis of a database of unique human and murine immunoglobulin heavy chain and light chain variable regions reveals that the precise patterns of exposed residues are different in human and murine antibodies, while most individual surface positions have strong preferences for a small number of residue types. Consideration of these surface patterns alone generates almost identical family groupings for light and heavy chain variable domain sequences to those produced by methods such as those of Kabat et al., where N-terminal framework sequences only are compared, or Tomlinson et al., in which entire variable region nucleotide sequences are used. This unexpected result suggests that the surfaces of V-regions are at least as well conserved as the core framework sequences. Furthermore, using these patterns of human and murine surface residues a novel method for the "humanization" of murine antibodies has been developed and tested.