Designed β-Boomerang Antiendotoxic and Antimicrobial Peptides: STRUCTURES AND ACTIVITIES IN LIPOPOLYSACCHARIDE*?

Lipopolysaccharide (LPS), an integral part of the outer membrane of Gram-negative bacteria, is involved in a variety of biological processes including inflammation, septic shock, and resistance to host-defense molecules. LPS also provides an environment for folding of outer membrane proteins. In this work, we describe the structure-activity correlation of a series of 12-residue peptides in LPS. NMR structures of the peptides derived in complex with LPS reveal boomerang-like β-strand conformations that are stabilized by intimate packing between the two aromatic residues located at the 4 and 9 positions. This structural feature renders these peptides with a high ability to neutralize endotoxicity, >80% at 10 nm concentration, of LPS. Replacements of these aromatic residues either with Ala or with Leu destabilizes the boomerang structure with the concomitant loss of antiendotoxic and antimicrobial activities. Furthermore, the aromatic packing stabilizing the β-boomerang structure in LPS is found to be maintained even in a truncated octapeptide, defining a structured LPS binding motif. The mode of action of the active designed peptides correlates well with their ability to perturb LPS micelle structures. Fourier transform infrared spectroscopy studies of the peptides delineate β-type conformations and immobilization of phosphate head groups of LPS. Trp fluorescence studies demonstrated selective interactions with LPS and the depth of insertion into the LPS bilayer. Our results demonstrate the requirement of LPS-specific structures of peptides for endotoxin neutralizations. In addition, we propose that structures of these peptides may be employed to design proteins for the outer membrane.LPS² or endotoxin, a major component of the outer leaflet of the outer membrane of Gram-negative bacteria, is critically involved in health and diseases of humans (1, 2). LPS is essential for bacterial survival through establishing an efficient permeability barrier against a variety of antimicrobial compounds including hydrophobic antibiotics, detergents, host-defense proteins, and antimicrobial peptides (3, 4). Several studies have demonstrated that LPS catalyzes folding of outer membrane proteins as a chaperone (5–7).LPS, a potent inducer of innate immune systems, hence called endotoxin, is primarily responsible for lethality in sepsis and septic shock syndromes associated with serious Gram-negative infections (8–10). Circulating LPS in bloodstream is intercepted by the phagocytic cells of the innate immune system. Once induced by LPS, these phagocytes produce proinflammatory cytokines, e.g. tumor necrosis factor-α, interleukin-6, and interleukin-1β, through the activation of a Toll-like pattern recognition receptor (11, 12). The release of cytokines in response to microbial invasion is a natural function of the innate immunity. However, an uncontrolled and overwhelming production of these cytokines may cause “endotoxic shock” or septic shock, typified by endothelial tissue damage, loss of vascular tone, coagulopathy, and multiple organ failure, often resulting in death (9, 10). Sepsis is the major cause of mortality in the intensive care unit, accounting for 200,000 deaths every year in the United States alone (13). It was demonstrated that release of LPS from antibiotic-treated Gram-negative bacteria can indeed enhance sepsis (14). Therefore, an effective antibiotic should not only exert antibacterial activities but also have the ability to sequester LPS and ameliorate its toxicity. Therefore, an amalgamated property of LPS-neutralizing and antimicrobial activity would be highly desirable for antimicrobial agents. Polymyxin B is a prototypical antimicrobial and antiendotoxic antibiotic; however, its neurotoxicity and nephrotoxicity limit its application to topical use (15). The increasing emergence of bacterial strains that are resistant to conventional antibiotics has initiated vital structure/function studies of membrane-perturbing cationic antimicrobial peptides (16–20). More recent studies have been conducted to understand interactions between antimicrobial peptides with LPS to gain insights into the mechanism of outer membrane perturbation, antibacterial activities, and LPS neutralization (21–26). These studies have delineated the role of amino acid sequence properties, LPS-peptide interactions by biophysical methods, and global structural parameters, obtained by CD and FTIR.Designing synthetic peptides and elucidation of three-dimensional structures in complex with LPS would be useful for the purpose of rational development of non-toxic antisepsis and antimicrobial therapeutics. Such studies will also be potentially instructive in establishing rules by which folded structures can be stabilized on the LPS surface. Extensive work in the field of peptide design primarily focuses on mimicking secondary structures and tertiary folds of proteins. Usually, short linear peptides are often structurally flexible; however, the functions of these peptides are highly dependent on their ability to adopt folded structures upon complex formation with their cognate receptors. In this regard, designed peptides that would yield high resolution structures in complex with LPS have not been well pursued. LPS, being a negatively charged amphiphilic molecule, interacts with naturally occurring peptides or protein fragments containing basic/polar and hydrophobic amino acids, although there are considerable variations in lengths, sequences, and amino acid compositions among these peptides (27, 28).Here, we have determined the three-dimensional structures of a series of 12-residue peptides in the context of LPS. To the best of our knowledge, these results show, for the first time, that atomic resolution structures of designed peptides obtained in LPS could be correlated with their antiendotoxic activities. Furthermore, the LPS-induced structures of active, inactive, and short peptide motif, presented here, may provide building blocks for the designing novel proteins for the outer membrane.     

Paleogenomics in a Temperate Environment: Shotgun Sequencing from an Extinct Mediterranean Caprine

Background

Numerous endemic mammals, including dwarf elephants, goats, hippos and deers, evolved in isolation in the Mediterranean islands during the Pliocene and Pleistocene. Most of them subsequently became extinct during the Holocene. Recently developed high-throughput sequencing technologies could provide a unique tool for retrieving genomic data from these extinct species, making it possible to study their evolutionary history and the genetic bases underlying their particular, sometimes unique, adaptations.

Methodology/Principals Findings

A DNA extraction of a ∼6,000 year-old bone sample from an extinct caprine (Myotragus balearicus) from the Balearic Islands in the Western Mediterranean, has been subjected to shotgun sequencing with the GS FLX 454 platform. Only 0.27% of the resulting sequences, identified from alignments with the cow genome and comprising 15,832 nucleotides, with an average length of 60 nucleotides, proved to be endogenous.

Conclusions

A phylogenetic tree generated with Myotragus sequences and those from other artiodactyls displays an identical topology to that generated from mitochondrial DNA data. Despite being in an unfavourable thermal environment, which explains the low yield of endogenous sequences, our study demonstrates that it is possible to obtain genomic data from extinct species from temperate regions.     

Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel

The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one α-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric α-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular α-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.     

Sustained swimming improves muscle growth and cellularity in gilthead sea bream

Two groups of juvenile gilthead sea bream were kept on two different swimming regimes (Exercise, E: 1.5 body length s⁻¹ or Control, C: voluntary activity) for 1 month. All fish were first adapted to an experimental diet low in protein and rich in digestible carbohydrates (37.2% protein, 40.4% carbohydrates, 12.5% lipid). The cellularity and capillarisation of white muscle from two selected areas (cranial (Cr), below the dorsal fin, and caudal (Ca), behind the anal fin) were compared. The body weight and specific growth rate (SGR) of group E rose significantly without an increment in feed intake, pointing to higher nutrient-use efficiency. The white muscle fibre cross-sectional area and the perimeter of cranial samples increased after sustained activity, evidencing that sustained exercise enhances hypertrophic muscle development. However, we cannot conclude or rule out the possibility of fibre recruitment because the experimental period was too short. In the control group, capillarisation, which is extremely low in gilthead sea bream white muscle, showed a significantly higher number of fibres with no surrounding capillaries (F0) in the cranial area than in the caudal area, unlike the exercise group. Sustained swimming improved muscle machinery even in tissue normally associated with short bouts of very rapid anaerobic activity. So, through its effect on the use of tissue reserves and nutrients, exercise contributes to improvements in fish growth what can contribute to reducing nitrogen losses.     

Radiation and phylogeography in the Japanese macaque, Macaca fuscata

The Japanese macaque (Macaca fuscata) presumably differentiated from eastern rhesus macaque (Macaca mulatta) populations during the Pleistocene and the two species are closely related. In order to analyse speciation and subspeciation events in the Japanese macaque and to describe historical and current relationships among their populations, we sequenced and analysed a fragment of 392bp of mitochondrial DNA (mtDNA) control region in 50 individuals belonging to six populations of Japanese macaque and compared these sequences with 89 eastern rhesus macaque control region sequences from GenBank/EMBL database. There were high genetic similarities between both species and only two positions were fixed within each species, which supports the inclusion of the Japanese macaque in a single species with eastern populations of rhesus macaques. Japanese macaque ancestors colonised Japan after the separation of the two species, estimated at between 0.31 and 0.88 million years ago (Mya). The star-like phylogeny, multimodal mismatch distribution, and lack of correlation between geographic and genetic distances are in accordance with a rapid dispersion of macaques throughout the archipelago after the arrival into Japan. The species shows low genetic variation within populations and high levels of genetic differentiation among populations with no mtDNA haplotype shared across populations. Genetic distances between Yakushima macaques (Macaca fuscata yakui) and any other population of Macaca fuscata fuscata subspecies are comparable to the distances between populations of Honshu, Awajishima, and Kyushu, not supporting the classification of Yakushima macaques as a different subspecies.     

Dietary Habits in Patients with Ischemic Stroke: A Case-Control Study

Background and Aims

Diet appears to have some role in stroke development. The objective of our study was to describe the dietary habits in patients admitted with acute ischemic stroke and compare selected dietary components with healthy controls. Adherence to healthy diet behaviors was also assessed.

Methods

A case-control study of consecutive patients with acute ischemic stroke admitted to the Neurology Department of Hospital del Mar from 2007 to 2010. Patients were matched by age and sex with control subjects. A previously validated nutritional survey was administered to patients and controls. Demographic data, vascular risk factors, caloric intake and dietary nutrients were evaluated. Intention to follow a healthy diet was also assessed in both groups.

Results

A total of 300 acute ischemic stroke patients and 300 controls with evaluation of dietary habits. No differences were observed in vascular risk factors, except smoking habit, diabetes and ischemic heart disease. Stroke patients reported a higher caloric intake: 2444.8(1736.8–3244.5) vs 2208.7(1753.1–2860.7) Kcal, p = 0.001. After adjusting for energy intake, patients had higher intake of proteins (p<0.001; OR 1.02), total cholesterol (p = 0.001; OR 1.04), and breaded foods (p = 0.001; OR 1.94) and lower consumption of probiotic yogurt (p = 0.002; OR 0.88). Compared to patients, control participants indicated greater intention to eat vegetables (p = 0.002; OR 1.5) and whole foods (p = 0.000; OR 2.4) and reduce their intake of salt (p = 0.002; OR 1.7), fat (p = 0.000; OR 3.7) and sweets (p = 0.004; OR 1.7) than patients.

Conclusion

We observed different dietary patterns between stroke patients and controls. Stroke patients have a higher caloric intake and are less concerned about maintaining healthy nutritional habits.