Search

The central nervous system (CNS) represents a site of sanctuary for many metastatic tumors when systemic therapies that control the primary tumor cannot effectively penetrate intracranial lesions. Non-small cell lung cancers (NSCLCs) are the most likely of all neoplasms to metastasize to the brain, with up to 60% of patients developing CNS metastases during the disease process. Targeted therapies such as tyrosine kinase inhibitors (TKIs) have helped reduce lung cancer mortality but vary considerably in their capacity to control CNS metastases. The ability of these therapies to effectively target lesions in the CNS depends on several of their pharmacokinetic properties, including blood-brain barrier permeability, affinity for efflux transporters, and binding affinity for both plasma and brain tissue. Despite the existence of numerous preclinical models with which to characterize these properties, many targeted therapies have not been rigorously tested for CNS penetration during the discovery process, whereas some made it through preclinical testing despite poor brain penetration kinetics. Several TKIs have now been engineered with the characteristics of CNS-penetrant drugs, with clinical trials proving these efforts fruitful. This Review outlines the extent and variability of preclinical evidence for the efficacy of NSCLC-targeted therapies, which have been approved by the US Food and Drug Administration (FDA) or are in development, for treating CNS metastases, and how these data correlate with clinical outcomes., Competing Interests: Competing interests A.J.P., J.D.H., T.S.Z. and I.O. report no potential conflicts of interest. K.W. and I.M. are employees of Taiho Pharmaceutical Company. R.S. has received research grants from Helsinn Healthcare SA, Loxo Oncology, Elevation Oncology and Merus, all unrelated to the current article., (© 2024. Published by The Company of Biologists Ltd.)

Technology is transforming societies worldwide. A major innovation is the emergence of robotics and autonomous systems (RAS), which have the potential to revolutionize cities for both people and nature. Nonetheless, the opportunities and challenges associated with RAS for urban ecosystems have yet to be considered systematically. Here, we report the findings of an online horizon scan involving 170 expert participants from 35 countries. We conclude that RAS are likely to transform land use, transport systems and human-nature interactions. The prioritized opportunities were primarily centred on the deployment of RAS for the monitoring and management of biodiversity and ecosystems. Fewer challenges were prioritized. Those that were emphasized concerns surrounding waste from unrecovered RAS, and the quality and interpretation of RAS-collected data. Although the future impacts of RAS for urban ecosystems are difficult to predict, examining potentially important developments early is essential if we are to avoid detrimental consequences but fully realize the benefits.

Background: A step change in the night environment is taking place, with the large-scale installation of bright, broad-spectrum road lighting such as white light-emitting diodes (LEDs). One justification for this is a reduction in road traffic collisions (RTCs). This study aimed to estimate the effect of new lighting on personal injury RTCs within a large UK city., Methods: We analysed a 9-year time series of weekly RTC personal injury counts in 132 areas of the city using multilevel modelling. The RTC rate over a full 24-hour period was the primary outcome; darkness and daylight RTC rates were secondary. The background change in RTC rate was separated from the change associated with the number of newly installed bright lamps by including a polynomial underlying time trend for the logarithm of the mean number of collisions per week for each area. The study was based on a rigorous, predesigned and archived protocol., Results: Within-area coefficients for the broad lighting effect were positive; as the number of bright lamps in an area increased, so did the RTC rate. The estimate for the increase in the within-area 24-hour RTC rate is 11% (95% CI 2% to 20%). The estimate of darkness-only RTCs is 16% (95% CI 2% to 32%). If the effect of lighting on darkness RTC rate is adjusted by that for daylight, one obtains 4% (95% CI -12% to +23%)., Conclusion: No evidence was found for bright lamps leading to an improvement in road safety in any of the analyses. For this city, introducing brighter road lighting may have compromised safety rather than reducing harm., Competing Interests: Competing interests: PM has previously published work questioning a large safety effect of road lighting., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY. Published by BMJ.)

In order to assess the colonization efficacy of the oral probiotic Streptococcus salivarius K12, a rapid method for specific detection and enumeration of the strain was developed. Here, we describe a two-step TaqMan™ quantitative PCR assay using primer-probe combinations targeting genes of the locus encoding the lantibiotic bacteriocin salivaricin B., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

More information on expected animal performance and carcass traits of forage-finished steers grazing warm-season annual forages is needed. To achieve this objective, a grazing trial was conducted in 2014, 2015, and 2016 (70, 63, and 56 d, respectively), with variation in length of grazing based on forage availability. Sixteen pastures (0.81 ha) were assigned to 1 of 4 forage treatments in a randomized complete block design. Forage treatments were brown midrib sorghum × sudangrass (BMR; Sorghum bicolor var. bicolor*bicolor var. sudanense ), sorghum × sudangrass (SS), pearl millet [PM; Pennisetum glaucum (L.)R.Br.], or pearl millet planted with crabgrass [PMCG; Digitaria sanguinalis (L.) Scop.]. Each year, British-cross beef steers ( n = 32; 3 y average: 429 ± 22 kg) were stratified by weight and randomly assigned to 1 of the 16 pastures for forage finishing. Each pasture was subdivided into two 0.405-ha paddocks for rotational stocking and a put-and-take stocking method was used to maintain a forage allowance of 116 kg forage dry matter/100 kg body weight (BW). Shrunk body weight and ultrasonically measured carcass composition were recorded at the initiation, middle, and end of each grazing season. Steers were harvested once forage availability became limited and chilled carcasses (24 h) were evaluated for yield grade and quality grade attributes. Statistical analysis was conducted using the GLIMMIX procedure in SAS 9.4 (Cary, NC) with main effects of treatment, year, and the interaction. Pasture and block were considered random effects while date was assessed as a main effect when applicable. Daily stocking densities were greater ( P < 0.04) for SS than PMCG in the first 20 d of 2014 and 2015. Forage treatment did not affect ( P > 0.17) total gain, total average daily gain, or body weight at any time point. Ultrasound composition traits of loin muscle area, 12th rib fat thickness, intramuscular fat, and rump fat were impacted ( P < 0.01) by scanning date. No differences ( P > 0.08) in forage treatments were observed for carcass characteristics associated with yield grade or quality grade. The findings suggest that forage-finished cattle during the summer months on BMR, SS, PM, and PMCG perform similarly, giving producers the option to use the most economical or practical forage type for their production system., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science.)

The demand for a year-round supply of fresh, locally grown, forage-finished beef products has created a need for forage-finishing strategies during the summer months in the southeast. A 3-yr study was conducted to evaluate four warm-season annual forages in a southeastern forage-finishing beef production system. Treatments were four forage species and included brown-midrib sorghum × sudangrass ( Sorghum bicolor var. bicolor*bicolor var. sudanense ; BMR), sorghum × sudangrass (SS), pearl millet [ Pennisetum glaucum (L.) R. Br.; PM], or pearl millet planted with crabgrass [ Digitaria sanguinalis (L.) Scop.; PMCG]. Treatments were distributed in a randomized complete block design with four replicates. Pastures (0.81 ha, experimental unit) were assigned to one of four forage treatments, subdivided, and rotationally stocked with a variable stocking density. British-cross beef steers ( n = 32; 3-yr average: 429 ± 22 kg) grazed for 70, 63, and 56 d in 2014, 2015, and 2016, respectively. Put-and-take animals were used to maintain a forage allowance of 116 kg forage dry matter /100 kg body weight. Forage mass was measured by clipping a 4.3-m 2 area in triplicate on d 0 and on 14-d intervals. Hand grab samples for forage nutritive value determination and quadrat clippings for species compositions were measured on d 0 and on 34-d intervals until termination of the trial. Forage mass was lowest ( P < 0.01) for PMCG at the initiation of the grazing trial, whereas BMR was greater ( P < 0.01) than SS at wk 6. Total digestible nutrients in 2014 were greater for SS compared to BMR and PM at the middle harvest ( P < 0.01) and BMR, PM, and PMCG at the final harvest ( P < 0.01). At the middle and final harvests in both 2015 and 2016, PM and PMCG contained greater ( P < 0.01) concentrations of crude protein than SS. These results suggest that BMR, SS, PM, and PMCG may all be used in southeastern forage-finishing beef production systems, as long as the producer strategically accounts for the slight growth and nutritive value differences throughout the season., (© The Author(s) 2019. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Background: Pneumococcal adherence to the nasopharyngeal epithelium is a critical step in colonisation and disease. The probiotic bacterium, Streptococcus salivarius, can inhibit pneumococcal adherence to epithelial cells in vitro. We investigated the mechanism(s) of inhibition using a human pharyngeal epithelial cell line (Detroit 562) following pre-administration of two different strains of S. salivarius., Results: Whilst the bacteriocin-encoding megaplasmids of S. salivarius strains K12 and M18 were essential to prevent pneumococcal growth on solid media, they were not required to inhibit pneumococcal adherence. Experiments testing S. salivarius K12 and two pneumococcal isolates (serotypes 19F and 6A) showed that inhibition of 19F may involve S. salivarius-mediated blocking of pneumococcal binding sites: a negative correlation was observed between adherence of K12 and 19F, and no inhibition occurred when K12 was prevented from contacting epithelial cells. K12-mediated inhibition of adherence by 6A may involve additional mechanisms, since no correlation was observed between adherence of K12 and 6A, and K12 could inhibit 6A adherence in the absence of cell contact., Conclusions: These results suggest that S. salivarius employs several mechanisms, including blocking pneumococcal binding sites, to reduce pneumococcal adherence to pharyngeal epithelial cells. These findings extend our understanding of how probiotics may inhibit pneumococcal adherence and could assist with the development of novel strategies to prevent pneumococcal colonisation in the future.

Streptococcus agalactiae (group B streptococcus [GBS]) colonizes the rectovaginal tract in 20% to 30% of women and during pregnancy can be transmitted to the newborn, causing severe invasive disease. Current routine screening and antibiotic prophylaxis have fallen short of complete prevention of GBS transmission, and GBS remains a leading cause of neonatal infection. We have investigated the ability of Streptococcus salivarius, a predominant member of the native human oral microbiota, to control GBS colonization. Comparison of the antibacterial activities of multiple S. salivarius strains by use of a deferred-antagonism test showed that S. salivarius strain K12 exhibited the broadest spectrum of activity against GBS. K12 effectively inhibited all GBS strains tested, including disease-implicated isolates from newborns and colonizing isolates from the vaginal tract of pregnant women. Inhibition was dependent on the presence of megaplasmid pSsal-K12, which encodes the bacteriocins salivaricin A and salivaricin B; however, in coculture experiments, GBS growth was impeded by K12 independently of the megaplasmid. We also demonstrated that K12 adheres to and invades human vaginal epithelial cells at levels comparable to GBS. Inhibitory activity of K12 was examined in vivo using a mouse model of GBS vaginal colonization. Mice colonized with GBS were treated vaginally with K12. K12 administration significantly reduced GBS vaginal colonization in comparison to nontreated controls, and this effect was partially dependent on the K12 megaplasmid. Our results suggest that K12 may have potential as a preventative therapy to control GBS vaginal colonization and thereby prevent its transmission to the neonate during pregnancy., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

As the global population urbanizes, dramatic changes are expected in city lighting and the urban form, which may threaten the functioning of urban ecosystems and the services they deliver. However, little is known about the ecological impact of lighting in different urban contexts. Movement is an important ecological process that can be disrupted by artificial lighting. We explored the impact of lighting on gap crossing for Pipistrellus pipistrellus, a species of bat (Chiroptera) common within UK cities. We aimed to determine whether the probability of crossing gaps in tree cover varied with crossing distance and lighting level, through stratified field surveys. We then used the resulting data on barrier thresholds to model the landscape resistance due to lighting across an entire city and explored the potential impact of scenarios for future changes to street lighting. The level of illumination required to create a barrier effect reduced as crossing distance increased. For those gaps where crossing was recorded, bats selected the darker parts of gaps. Heavily built parts of the case study city were associated with large and brightly lit gaps, and spatial models indicate movement would be highly restricted in these areas. Under a scenario for brighter street lighting, the area of accessible land cover was further reduced in heavily built parts of the city. We believe that this is the first study to demonstrate how lighting may create resistance to species movement throughout an entire city. That connectivity in urban areas is being disrupted for a relatively common species raises questions about the impacts on less tolerant groups and the resilience of bat communities in urban centres. However, this mechanistic approach raises the possibility that some ecological function could be restored in these areas through the strategic dimming of lighting and narrowing of gaps., (© 2015 The Authors. Global Change Biology Published by John Wiley & Sons Ltd.)

Bacteriocin-producing probiotic Streptococcus salivarius M18 offers beneficial modulatory capabilities within the oral microbiome, apparently through potent inhibitory activity against potentially deleterious bacteria, such as Streptococcus pyogenes. The oral cavity persistence of S. salivarius M18 was investigated in 75 subjects receiving four different doses for 28 days. Sixty per cent of the subjects already had some inhibitor-producing S. salivarius in their saliva prior to probiotic intervention. Strain M18's persistence was dependent upon the dose, but not the period of administration. Culture analysis indicated that in some individuals the introduced strain had almost entirely replaced the indigenous S. salivarius, though the total numbers of the species did not increase. Selected subjects showing either high or low probiotic persistence had their salivary populations profiled using Illumina sequencing of the V6 region of the 16S rRNA gene. Analysis indicated that while certain bacterial phenotypes were markedly modulated, the overall composition of the oral microbiome was not modified by the probiotic treatment. Megaplasmids encoding bacteriocins and adhesion factors were transferred in vitro to generate a transconjugant S. salivarius exhibiting enhanced antimicrobial production and binding capabilities to HEp-2 cells. Since no widespread perturbation of the existing indigenous microbiota was associated with oral instillation and given its antimicrobial activity against potentially pathogenic streptococci, it appears that application of probiotic strain M18 offers potential low impact alternative to classical antibiotic prophylaxis. For candidate probiotic strains having relatively poor antimicrobial or adhesive properties, unique derivatives displaying improved probiotic performance may be engineered in vitro by megaplasmid transfer.

Artificial lighting is strongly associated with urbanisation and is increasing in its extent, brightness and spectral range. Changes in urban lighting have both positive and negative effects on city performance, yet little is known about how its character and magnitude vary across the urban landscape. A major barrier to related research, planning and governance has been the lack of lighting data at the city extent, particularly at a fine spatial resolution. Our aims were therefore to capture such data using aerial night photography and to undertake a case study of urban lighting. We present the finest scale multi-spectral lighting dataset available for an entire city and explore how lighting metrics vary with built density and land-use. We found positive relationships between artificial lighting indicators and built density at coarse spatial scales, whilst at a local level lighting varied with land-use. Manufacturing and housing are the primary land-use zones responsible for the city's brightly lit areas, yet manufacturing sites are relatively rare within the city. Our data suggests that efforts to address light pollution should broaden their focus from residential street lighting to include security lighting within manufacturing areas.

Considerable human illness can be linked to the development of oral microbiota disequilibria. The predominant oral cavity commensal, Streptococcus salivarius has emerged as an important source of safe and efficacious probiotics, capable of fostering more balanced, health-associated oral microbiota. Strain K12, the prototype S. salivarius probiotic, originally introduced to counter Streptococcus pyogenes infections, now has an expanded repertoire of health-promoting applications. K12 and several more recently proposed S. salivarius probiotics are now being applied to control diverse bacterial consortia infections including otitis media, halitosis and dental caries. Other potential applications include upregulation of immunological defenses against respiratory viral infections and treatment of oral candidosis. An overview of the key steps required for probiotic development is also presented.

Background: Urbanization is characterized by high levels of sealed land-cover, and small, geometrically complex, fragmented land-use patches. The extent and density of urbanized land-use is increasing, with implications for habitat quality, connectivity and city ecology. Little is known about densification thresholds for urban ecosystem function, and the response of mammals, nocturnal and cryptic taxa are poorly studied in this respect. Bats (Chiroptera) are sensitive to changing urban form at a species, guild and community level, so are ideal model organisms for analyses of this nature., Methodology/principal Findings: We surveyed bats around urban ponds in the West Midlands conurbation, United Kingdom (UK). Sites were stratified between five urban land classes, representing a gradient of built land-cover at the 1 km(2) scale. Models for bat presence and activity were developed using land-cover and land-use data from multiple radii around each pond. Structural connectivity of tree networks was used as an indicator of the functional connectivity between habitats. All species were sensitive to measures of urban density. Some were also sensitive to landscape composition and structural connectivity at different spatial scales. These results represent new findings for an urban area. The activity of Pipistrellus pipistrellus (Schreber 1774) exhibited a non-linear relationship with the area of built land-cover, being much reduced beyond the threshold of ∼60% built surface. The presence of tree networks appears to mitigate the negative effects of urbanization for this species., Conclusions/significance: Our results suggest that increasing urban density negatively impacts the study species. This has implications for infill development policy, built density targets and the compact city debate. Bats were also sensitive to the composition and structure of the urban form at a range of spatial scales, with implications for land-use planning and management. Protecting and establishing tree networks may improve the resilience of some bat populations to urban densification.

Bac8c (RIWVIWRR-NH(2)) is an 8-amino-acid peptide derived from Bac2A (RLARIVVIRVAR-NH(2)), a C3A/C11A variant of the naturally occurring bovine peptide, bactenecin (also known as bovine dodecapeptide), the smallest peptide with activity against a range of pathogenic Gram-positive and Gram-negative bacteria, as well as yeast. The effects of Bac8c on Escherichia coli were examined by studying its bacteriostatic and bactericidal properties, demonstrating its effects on proton motive force generation, and visually analyzing (via transmission electron microscopy) its effects on cells at different concentrations, in order to probe the complexities of the mechanism of action of Bac8c. Results were consistent with a two-stage model for the Bac8c mode of action. At sublethal concentrations (3 μg/ml), Bac8c addition resulted in transient membrane destabilization and metabolic imbalances, which appeared to be linked to inhibition of respiratory function. Although sublethal concentrations resulted in deleterious downstream events, such as methylglyoxal formation and free radical generation, native E. coli defense systems were sufficient for full recovery within 2 h. In contrast, at the minimal bactericidal concentration (6 μg/ml), Bac8c substantially but incompletely depolarized the cytoplasmic membrane within 5 min and disrupted electron transport, which in turn resulted in partial membrane permeabilization and cell death.

For cationic antimicrobial peptides to become useful therapeutic agents, it is important to understand their mechanism of action. To obtain high resolution data, this involves studying the structure and membrane interaction of these peptides in tractable model bacterial membranes rather than directly utilizing more complex bacterial surfaces. A number of lipid mixtures have been used as bacterial mimetics, including a range of lipid headgroups, and different ratios of neutral to negatively charged headgroups. Here we examine how the structure and membrane interaction of aurein 2.2 and some of its variants depend on the choice of lipids, and how these models correlate with activity data in intact bacteria (MICs, membrane depolarization). Specifically, we investigated the structure and membrane interaction of aurein 2.2 and aurein 2.3 in 1:1 cardiolipin/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (CL/POPG) (mol/mol), as an alternative to 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POPG and a potential model for Gram positive bacteria such as S. aureus. The structure and membrane interaction of aurein 2.2, aurein 2.3, and five variants of aurein 2.2 were also investigated in 1:1 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE)/POPG (mol/mol) lipids as a possible model for other Gram positive bacteria, such as Bacillus cereus. Solution circular dichroism (CD) results demonstrated that the aurein peptides adopted α-helical structure in all lipid membranes examined, but demonstrated a greater helical content in the presence of POPE/POPG membranes. Oriented CD and ³¹P NMR results showed that the aurein peptides had similar membrane insertion profiles and headgroup disordering effects on POPC/POPG and CL/POPG bilayers, but demonstrated reduced membrane insertion and decreased headgroup disordering on mixing with POPE/POPG bilayers at low peptide concentrations. Since the aurein peptides behaved very differently in POPE/POPG membrane, minimal inhibitory concentrations (MICs) of the aurein peptides in B. cereus strain C737 were determined. The MIC results indicated that all aurein peptides are significantly less active against B. cereus than against S. aureus and S. epidermidis. Overall, the data suggest that it is important to use a relevant model for bacterial membranes to gain insight into the mode of action of a given antimicrobial peptide in specific bacteria., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: The potential for reduced pollination ecosystem service due to global declines of bees and other pollinators is cause for considerable concern. Habitat degradation, destruction and fragmentation due to agricultural intensification have historically been the main causes of this pollinator decline. However, despite increasing and accelerating levels of global urbanization, very little research has investigated the effects of urbanization on pollinator assemblages. We assessed changes in the diversity, abundance and species composition of bee and hoverfly pollinator assemblages in urban, suburban, and rural sites across a UK city., Methodology/principal Findings: Bees and hoverflies were trapped and netted at 24 sites of similar habitat character (churchyards and cemeteries) that varied in position along a gradient of urbanization. Local habitat quality (altitude, shelter from wind, diversity and abundance of flowers), and the broader-scale degree of urbanization (e.g. percentage of built landscape and gardens within 100 m, 250 m, 500 m, 1 km, and 2.5 km of the site) were assessed for each study site. The diversity and abundance of pollinators were both significantly negatively associated with higher levels of urbanization. Assemblage composition changed along the urbanization gradient with some species positively associated with urban and suburban land-use, but more species negatively so. Pollinator assemblages were positively affected by good site habitat quality, in particular the availability of flowering plants., Conclusions/significance: Our results show that urban areas can support diverse pollinator assemblages, but that this capacity is strongly affected by local habitat quality. Nonetheless, in both urban and suburban areas of the city the assemblages had fewer individuals and lower diversity than similar rural habitats. The unique development histories of different urban areas, and the difficulty of assessing mobile pollinator assemblages in just part of their range, mean that complementary studies in different cities and urban habitats are required to discover if these findings are more widely applicable.

Infections caused by multidrug-resistant (MDR) Gram-negative bacteria represent a major global health problem. Polymyxin antibiotics such as colistin have resurfaced as effective last-resort antimicrobials for use against MDR Gram-negative pathogens, including Acinetobacter baumannii. Here we show that A. baumannii can rapidly develop resistance to polymyxin antibiotics by complete loss of the initial binding target, the lipid A component of lipopolysaccharide (LPS), which has long been considered to be essential for the viability of Gram-negative bacteria. We characterized 13 independent colistin-resistant derivatives of A. baumannii type strain ATCC 19606 and showed that all contained mutations within one of the first three genes of the lipid A biosynthesis pathway: lpxA, lpxC, and lpxD. All of these mutations resulted in the complete loss of LPS production. Furthermore, we showed that loss of LPS occurs in a colistin-resistant clinical isolate of A. baumannii. This is the first report of a spontaneously occurring, lipopolysaccharide-deficient, Gram-negative bacterium.

Previous studies on aurein 2.2 and 2.3 in DMPC/DMPG and POPC/POPG membranes have shown that bilayer thickness and phosphatidylglycerol content have a significant impact on the interaction of these peptides with membrane bilayers. Further examination with the DiSC(3)5 assay has indicated that aurein 2.2 induces greater membrane leakage than aurein 2.3 in Staphylococcus aureus C622. The only difference between these peptides is a Leu to Ile mutation at residue 13. To better understand the importance of this residue, the structure and activity of the L13A, L13F, and L13V mutants were investigated. In addition, we investigated a number of peptides with truncations at the C-terminus to determine whether the C-terminus, which contains residue 13, is crucial for antimicrobial activity. Solution circular dichroism results demonstrated that the L13F mutation and the truncation of the C-terminus by six residues resulted in decreased helical content, whereas the L13A or L13V mutation and the truncation of the C-terminus by three residues showed little to no effect on the structure. Oriented circular dichroism results demonstrated that only an extensive C-terminal truncation reduced the ability of the peptide to insert into lipid bilayers. (31)P NMR spectroscopy showed that all peptides disorder the headgroups. The implications of these results in terms of antimicrobial activity and the ability of these peptides to induce leakage in S. aureus are discussed. The results suggest that the presence of the 13th residue in aurein 2.2 is important for structure and activity, but the exact nature of residue 13 is less important as long as it is a hydrophobic residue., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The effects of hydrophobic thickness and the molar phosphatidylglycerol (PG) content of lipid bilayers on the structure and membrane interaction of three cationic antimicrobial peptides were examined: aurein 2.2, aurein 2.3 (almost identical to aurein 2.2, except for a point mutation at residue 13), and a carboxy C-terminal analog of aurein 2.3. Circular dichroism results indicated that all three peptides adopt an alpha-helical structure in the presence of a 3:1 molar mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DMPC/DMPG), and 1:1 and 3:1 molar mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPC/POPG). Oriented circular dichroism data for three different lipid compositions showed that all three peptides were surface-adsorbed at low peptide concentrations, but were inserted into the membrane at higher peptide concentrations. The (31)P solid-state NMR data of the three peptides in the DMPC/DMPG and POPC/POPG bilayers showed that all three peptides significantly perturbed lipid headgroups, in a peptide or lipid composition-dependent manner. Differential scanning calorimetry results demonstrated that both amidated aurein peptides perturbed the overall phase structure of DMPC/DMPG bilayers, but perturbed the POPC/POPG chains less. The nature of the perturbation of DMPC/DMPG bilayers was most likely micellization, and for the POPC/POPG bilayers, distorted toroidal pores or localized membrane aggregate formation. Calcein release assay results showed that aurein peptide-induced membrane leakage was more severe in DMPC/DMPG liposomes than in POPC/POPG liposomes, and that aurein 2.2 induced higher calcein release than aurein 2.3 and aurein 2.3-COOH from 1:1 and 3:1 POPC/POPG liposomes. Finally, DiSC(3)5 assay data further delineated aurein 2.2 from the others by showing that it perturbed the lipid membranes of intact S. aureus C622 most efficiently, whereas aurein 2.3 had the same efficiency as gramicidin S, and aurein 2.3-COOH was the least efficient. Taken together, these data show that the membrane interactions of aurein peptides are affected by the hydrophobic thickness of the lipid bilayers and the PG content.

We utilized a series of analogs of D-V13K (a 26-residue amphipathic alpha-helical antimicrobial peptide, denoted D1) to compare and contrast the role of hydrophobicity on antifungal and antibacterial activity to the results obtained previously with Pseudomonas aeruginosa strains. Antifungal activity for zygomycota fungi decreased with increasing hydrophobicity (D-V13K/A12L/A20L/A23L, denoted D4, the most hydrophobic analog was sixfold less active than D1, the least hydrophobic analog). In contrast, antifungal activity for ascomycota fungi increased with increasing hydrophobicity (D4, the most hydrophobic analog was fivefold more active than D1). Hemolytic activity is dramatically affected by increasing hydrophobicity with peptide D4 being 286-fold more hemolytic than peptide D1. The therapeutic index for peptide D1 is 1569-fold and 62-fold better for zygomycota fungi and ascomycota fungi, respectively, compared with peptide D4. To reduce the hemolytic activity of peptide D4 and improve/maintain the antifungal activity of D4, we substituted another lysine residue in the center of the non-polar face (V16K) to generate D5 (D-V13K/V16K/A12L/A20L/A23L). This analog D5 decreased hemolytic activity by 13-fold, enhanced antifungal activity to zygomycota fungi by 16-fold and improved the therapeutic index by 201-fold compared with D4 and represents a unique approach to control specificity while maintaining high hydrophobicity in the two hydrophobic segments on the non-polar face of D5.

A synchrotron-based x-ray photoemission electron microscope (X-PEEM) was used to investigate the coadsorption of a mixture of human albumin serum and SUB-6, a synthetic antimicrobial peptide, to a phase-segregated polystyrene/poly(methyl methacrylate) (PMMA) substrate at varying concentrations and pH. The authors show that X-PEEM could detect the peptide adsorbed from solution at concentrations as low as 5.5 x 10(-9)M and could differentiate the four components via near-edge x-ray absorption fine structure spectromicroscopy. At neutral pH the SUB-6 peptide adsorbed preferentially to PMMA. At a pH of 11.8 where the charge on the peptide was neutralized, there was a more balanced adsorption of both species on the PMMA domains. The authors interpret these observations as indicative of the formation of an electrostatic complex between positive peptide and negative protein at pH of 7.0. This solution complex had an adsorption behavior that depended on the polarity of the substrate domains, and favored adsorption to the electronegative PMMA regions. At a pH of 11.8 the complex formation was suppressed and a more competitive adsorption process was observed.

In our previous study, we utilized a 26-residue amphipathic alpha-helical antimicrobial peptide L-V13K (Chen et al., Antimicrob Agents Chemother 2007, 51, 1398-1406) as the framework to study the effects of peptide hydrophobicity on the mechanism of its antimicrobial action. In this study, we explored the effects of net charge and the number of positively charged residues on the hydrophilic/polar face of L-V13K on its biological activity (antimicrobial and hemolytic) and biophysical properties (hydrophobicity, amphipathicity, helicity, and peptide self-association). The net charge of V13K analogs at pH 7 varied between -5 and +10 and the number of positively charged residues varied from 1 to 10. The minimal inhibitory concentrations (MIC) against six strains of Pseudomonas aeruginosa as well as other gram-negative and gram-positive bacteria were determined along with the maximal peptide concentration that produces no hemolysis of human red blood cells (MHC). Our results show that the number of positively charged residues on the polar face and net charge are both important for both antimicrobial activity and hemolytic activity. The most dramatic observation is the sharp transition of hemolytic activity on increasing one positive charge on the polar face of V13K i.e., the change from +8 to +9 resulted in greater than 32-fold increase in hemolytic activity (250 microg/ml to <7.8 microg/ml, respectively)., ((c) 2007 Wiley Periodicals, Inc.)

Cationic antimicrobial peptides are a novel type of antibiotic offering much potential in the treatment of microbial-related diseases. They offer many advantages for commercial development, including a broad spectrum of action and modest size. However, despite the identification or synthetic production of thousands of such peptides, the mode of action remains elusive, except for a few examples. While the dogma for the mechanism of action of antimicrobial peptides against bacteria is believed to be through pore formation or membrane barrier disruption, some peptides clearly act differently and other intracellular target sites have been identified. This article presents an updated review of how cationic antimicrobial peptides are able to affect bacterial killing, with a focus on internal targets.

The intrinsic variation in the near-edge X-ray absorption fine structure (NEXAFS) spectra of peptides and proteins provide an opportunity to identify and map them in various biological environments, without additional labeling. In principle, with sufficiently accurate spectra, peptides (<50 amino acids) or proteins with unusual sequences (e.g., cysteine- or methionine-rich) should be differentiable from other proteins, since the NEXAFS spectrum of each amino acid is distinct. To evaluate the potential for this approach, we have developed X-SpecSim, a tool for quantitatively predicting the C, N, and O 1s NEXAFS spectra of peptides and proteins from their sequences. Here we present the methodology for predicting such spectra, along with tests of its precision using comparisons to the spectra of various proteins and peptides. The C 1s, N 1s, and O 1s spectra of two novel antimicrobial peptides, Indolicidin (ILPWKWPWWPWRR-NH2) and Sub6 (RWWKIWVIRWWR-NH2), as well as human serum albumin and fibrinogen are reported and interpreted. The ability to identify, differentiate, and quantitatively map an antimicrobial peptide against a background of protein is demonstrated by a scanning transmission X-ray microscopy study of a mixture of albumin and sub6.

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic mutacin activity. In this study, bioinformatic and mutational analyses were employed to demonstrate that the antimicrobial repertoire of strain UA159 includes mutacin IV (specified by the nlm locus) and a newly identified bacteriocin, mutacin V (encoded by SMU.1914c).

Streptococcus mutans UA159, the genome sequence reference strain, exhibits nonlantibiotic bacteriocin (mutacin) activity. In this study, we have combined bioinformatic and mutational analyses to identify the ABC transporter designated NlmTE, which is required for mutacin biogenesis in strain UA159 as well as in another mutacin producer, S. mutans N.

Background & Objectives: The mutans streptococci (MS) are a group of 7 species of dental cariesassociated bacteria of which Streptococcus mutans and Streptococcus sobrinus are the most important in humans. Many MS produce bacteriocin-like inhibitory substances (BLIS), some of which have been characterised as small peptides capable of inhibiting the growth of closely-related species. These peptides have most commonly been referred to as mutacins. S. mutans strains N and UA140 appear to have closely similar BLIS activities. Both produce mutacins that seem to target the same species of bacteria. On closer analysis however, these two strains have been shown to produce distinctly different mutacins, known as mutacin N and mutacin I respectively. In the present study the mutacin N structural gene (mutN) was cloned and compared with the mutacin I structural gene (mutA)., Methods: Cloning and sequencing of S. mutans N was done. The distribution of mutN using DNA from 216 streptococcal strains was determined by dot blotting., Results: Mut N was cloned and sequenced from an 1800 bp Bam HI/Eco RI fragment. PCR with the mutN primers mutNF and mutNR on the four mutN-positive strains identified identical bands to S. mutans N. The location of mutN differs significantly from that of mutA in that it is directly upstream of comC, a gene encoding a putative competence stimulating factor., Interpretation & Conclusion: The close upstream proximity of mutN to comC suggests a link between mutacin N production and competence development. Further studies need to be done to detect competence-related genes in S. mutans strain N.

It is possible to acquire a truncated echo, in which part of the information at the beginning or end of the echo is missing, and to restore the missing information by conjugation. This process was shown by means of edge spread function in phantoms and brain images in volunteers to introduce little, if any, degradation of image quality and can be used to improve sequence efficiency or to shorten echo time.

To improve the quality of projection angiograms generated from three-dimensional magnetic resonance (MR) angiography data, the authors applied voxel shifting to create intermediate sections ("section doubling") prior to maximum intensity projection. To date, the authors have processed MR angiography studies with and without section doubling in 20 cases. Section doubling resulted in improved vessel contrast and delineation of continuity (especially of small vessels) in all cases.

Misregistration between section position and a feature of interest, such as a lesion, can result in loss of contrast on magnetic resonance images. Because the section direction typically has the poorest resolution, reduction of section misregistration can improve lesion contrast. Lesions occur at random sites; hence, to reduce misregistration, it is necessary to repeat imaging with a small amount of offset. This is impractical. Therefore, a reconstruction algorithm that yields offset sections was implemented, in which sections were produced with a zero- and a half-section shift. This algorithm was tested in 14 patients with multiple sclerosis. As expected, because of randomness associated with section placement relative to lesions, the half-shifted sections provided greater contrast as often as the zero-shifted sections. The combined set improved contrast significantly in 39% of lesions; improvement was greatest in lesions with lesser contrast. Signal-to-noise ratios were not affected by this process, which appears to be useful in magnetic resonance image reconstruction.

Multisection, dual-echo magnetic resonance (MR) transaxial images of blood vessels contain both anatomic and qualitative information about flow. Even so, the images are produced as a series of two-dimensional tomographic sections from which full visualization of connected structures is difficult. A computer algorithm was developed that automatically detects flowing blood based on pixel intensity and calculated T2 and provides reconstructed views of vessels while analyzing and displaying flow characteristics. Images of abdominal vessels, aortic aneurysms, and the heart were encoded by flow and color to demonstrate depth. In addition, these data were reconstructed to derive a more accurate assessment of patency. With this technique, transaxial images can be used to analyze flow patterns, determine patent areas, and visualize all levels of vessels in a single image.

Magnetic resonance imaging of the cardiovascular system offers great promise in the detection and characterization of the anatomic, physiologic, and biochemical consequences of atherosclerosis. This review will focus on the potential applications of MRI for evaluating atherosclerosis of the abdominal aorta and iliofemoral vessels.

The capability of magnetic resonance (MR) imaging for detecting aortic, iliac, and femoral stenoses and occlusions was evaluated. Multisection spin-echo studies at 0.35 tesla were obtained of the infrarenal aorta to the femoral bifurcation in 24 patients, all of whom had undergone intraarterial angiography within 14 days of imaging. Transaxial MR images were compared with the angiograms. Arterial stenoses and occlusions in these vessels detected by MR imaging correlated with angiographic findings in 91% of the instances. Protrusional atherosclerotic plaques and occlusions and stenoses in the aortoiliac region were demonstrated accurately on MR images; complications of previous vascular surgery, such as aneurysms at sites of previous anastomoses or endarterectomy, were also identified. Due to the limited spatial resolution, MR images failed to demonstrate some femoral stenoses. MR imaging may be used for evaluation of aortoiliac vascular disease and for follow-up study after surgical revascularization. However, the limited spatial resolution, noncomposite display of the aortoiliofemoral circulation, and lack of evaluation of peripheral runoff provided by current MR imaging techniques militate against its replacing angiography prior to vascular intervention.

The unique properties of magnetic resonance imaging result in the potential to differentiate various components of the diseased arterial wall. In this article four cases are presented in which magnetic resonance imaging showed mural aortic thrombus and its anatomic relationship to the visceral and renal arteries. Once thrombus is identified and localized specific operative strategies can be undertaken to prevent recurrent embolic events and/or avoid perioperative thromboembolic complications.

Conjugation can be used to synthesize half of the data acquired during a conventional two-dimensional Fourier transform imaging procedure, thus reducing imaging time by nearly half. The images acquired by this process have the same object contrast and spatial resolution as conventional images do, but with a 40% reduction in the signal-to-noise ratio (S/N). Conjugation can be used to advantage in magnetic resonance imaging units in which S/N levels are higher than needed to permit imaging with a single acquisition of each projection.

Longitudinal vascular structures are difficult to observe on the standard abdominal transaxial magnetic resonance (MR) image sections. To display the information in a three-dimensional reconstruction, an algorithm was written to identify blood flow in a series of transaxial MR sections and was applied to reconstructing images of the aorta and iliac arteries in 12 patients with aortic aneurysm, dissection, or aortoiliac atherosclerosis. Results were validated by angiography. In all patients, the outline of the flow channel in the reconstructed image followed closely the outline of the lumen on angiograms. In aortic dissection, the MR images showed the two lumens more completely than did the angiograms, and in atherosclerosis, sites of vascular stenosis were correctly identified on MR images. The technique is valuable in providing anatomic information as well as functional information on cross-sectional areas and relative flow velocities.

A computer program was written to detect blood flow in a sequence of magnetic resonance images, using reduction in first echo intensity and prolongation of calculated T2 value, and to construct three-dimensional vessel maps from the detected flow regions. Reconstructed vessel images were coded to show cross-sectional area of the lumen and relative flow velocity along the length of the vessel. The program was applied to imaging the aorta and iliac arteries in 11 patients with atherosclerosis and results were compared with angiography. All sites of narrowing were identified by the algorithm, but no flow at all was detected at one site of partial occlusion.

Phase-sensitive imaging was used to correlate signal distribution with phase shift and velocity distribution in spin-echo magnetic resonance imaging (MRI). Flow-dependent, changing intensity patterns that were seen in a constant-flow phantom study were explained by the simultaneous effects of inflow signal enhancement, first-echo dephasing, and outflow signal loss occurring during laminar flow. In clinical studies, first-echo dephasing was shown during laminar flow in the inferior vena cava. Turbulent flow was demonstrated in the descending thoracic aorta during late systolic flow, and turbulent dephasing-rephasing was shown in the abdominal aorta.

Practical constraints make it difficult to build large-aperture echo-planar magnetic resonance (MR) imagers. The implementation of a pediatric imager and its performance are described. Spatial resolution and signal-to-noise levels comparable to those of 1982 state-of-the-art MR imagers have been achieved in imaging times of 0.05-0.15 seconds. T1 and T2 information are obtainable in the echo-planar mode. A major issue is that of chemical-shift displacements.