Poly-ε-Lysine or Mel4 Antimicrobial Surface Modification on a Novel Peptide Hydrogel Bandage Contact Lens

Microbial keratitis (MK) is a serious issue in many countries and is often caused by contact lens wear. Antimicrobial peptides (AMPs) are a potentially useful tool for creating antimicrobial surfaces in light of increasing antibiotic resistance. Poly-ε-lysine (pεK) is an AMP that has been used extensively as a food preservative and Mel4 has recently been synthesized and studied as an antimicrobial coating for contact lenses. A hydrogel synthesized of pεK cross-linked with biscarboxylic acids provides a potential lens material which has many surface free amines, that can be subsequently used to attach additional AMPs, creating an antimicrobial lens. The aim of this study is to investigate pεK hydrogels against a clinical strain of Pseudomonas aeruginosa (P. aeruginosa) for preventing or treating MK. Covalent attachment of AMPs is investigated and confirmed by fluorescently tagged peptides. Bound pεK effectively reduces the number of adherent P. aeruginosa in vitro (>3 log). In ex vivo studies positive antimicrobial activity is observed on bare pεK hydrogels and those with additionally bound pεK or Mel4; lenses allow the maintenance of the corneal epithelium. A pεK hydrogel contact lens with additional AMPs can be a therapeutic tool to reduce the incidence of MK

Kinetic analysis of copper(I)/feringa-phosphoramidite catalysed AlEt3 1,4-addition to cyclohex-2-en-1-one

ReactIR studies of mixtures of AlEt3 (A) and cyclohex-2-en-1-one (CX) in Et2O indicate immediate formation of the Lewis acid-base complex (CX.A) at -40 oC (K = 12.0 M-1, ΔGo react -1.1 kcal mol-1). Copper(I) catalysts, derived from pre-catalytic Cu(OAc)2 (up to 5 mol- %) and (R,S,S)-P(binaphtholate){N(CHMePh)2} [Feringa’s ligand (L), up to 5 mol-%] convert CX.A (0.04-0.3 M) into its 1,4-addition product enolate (E) within 2000 sec at -40 oC. Kinetic studies (ReactIR and chiral GC) of CX.A, CX and (R)-3-ethylcyclohexanone (P, the H+ quench product of enolate E) show that the true catalyst is formed in the first 300 sec and this subsequently provides P in 82% ee. This true catalyst converts CX.A to E with a rate law [Cu]1.5[L]0.66[CX.A]1 when [L]/[Cu] ≤ 3.5. Above this ligand ratio inhibition by added ligand with order [L]-2.5 is observed. A rate determining step (rds) of Cu3L2(CX.A)2 stoichiometry is shown to be most consistent with the rate law. The presence of the enolate in the active catalyst (Graphical Abstract) best accounts for the reaction’s induction period and molecularity as [E] ≡ [CX.A]. Catalysis proceeds through a ‘shuttling mechanism’ between two C2 symmetry related ground state intermediates. Each turnover consumes one equivalent of CX.A, expels one molecule of E and forms the new Cu-Et bond needed for the next cycle (Graphic Abstract). The observed ligand (L) inhibition and a non-linear ligand Lee effect on the ee of P are all well simulated by the kinetic model. DFT studies [ωB97X-D/SRSC] support coordination of CX.A to the groundstate Cu-trimer and its rapid conversion to E

Domestication as innovation : the entanglement of techniques, technology and chance in the domestication of cereal crops

The origins of agriculture involved pathways of domestication in which human behaviours and plant genetic adaptations were entangled. These changes resulted in consequences that were unintended at the start of the process. This paper highlights some of the key innovations in human behaviours, such as soil preparation, harvesting and threshing, and how these were coupled with genetic ‘innovations’ within plant populations. We identify a number of ‘traps’ for early cultivators, including the needs for extra labour expenditure on crop-processing and soil fertility maintenance, but also linked gains in terms of potential crop yields. Compilations of quantitative data across a few different crops for the traits of nonshattering and seed size are discussed in terms of the apparently slow process of domestication, and parallels and differences between different regional pathways are identified. We highlight the need to bridge the gap between a Neolithic archaeobotanical focus on domestication and a focus of later periods on crop-processing activities and labour organization. In addition, archaeobotanical data provide a basis for rethinking previous assumptions about how plant genetic data should be related to the origins of agriculture and we contrast two alternative hypotheses: gradual evolution with low selection pressure versus metastable equilibrium that prolonged the persistence of ‘semi-domesticated’ populations. Our revised understanding of the innovations involved in plant domestication highlight the need for new approaches to collecting, modelling and integrating genetic data and archaeobotanical evidence

Electrostatic and Covalent Binding of an Antibacterial Polymer to Hydroxyapatite for Protection against Escherichia coli Colonization

Orthopedic-device-related infections are notorious for causing physical and psychological trauma to patients suffering from them. Traditional methods of treating these infections have relied heavily on antibiotics and are becoming ineffectual due to the rise of antibiotic-resistant bacteria. Mimics of antimicrobial peptides have emerged as exciting alternatives due to their favorable antibacterial properties and lack of propensity for generating resistant bacteria. In this study, the efficacy of an antibacterial polymer as a coating material for hydroxyapatite and glass surfaces, two materials with wide ranging application in orthopedics and the biomedical sciences, is demonstrated. Both physical and covalent modes of attachment of the polymer to these materials were explored. Polymer attachment to the material surfaces was confirmed via X-ray photoelectron spectroscopy and contact angle measurements. The modified surfaces exhibited significant antibacterial activity against the Gram-negative bacterium E. coli, and the activity was retained for a prolonged period on the surfaces of the covalently modified materials

Mechanistic-insight-driven rate enhancement of asymmetric copper-catalyzed 1,4-addition of dialkylzinc reagents to enones

The combination of [Cu(MeCN)4]TFA·TFAH (TFA = O2CCF3) with Feringa’s phosphoramidite ligand (LA) provides an exceptionally active (0.75 mol %) catalyst for asymmetric conjugate additions of ZnR2 (R = Et and Me at −40 to −80 °C) to enones. Kinetic and other studies of the addition of ZnEt2 to cyclohex-2-en-1-one indicate a transition state stoichiometry composition of (ZnEt2)3(enone)4Cu2(LA)3 that is generated by transmetalation from Et2Zn(enone)2. Catalyst genesis is significantly slower than turnover (which has limited previous attempts to attain useful kinetic data); in the initial stages, varying populations of catalytically inactive, off-cycle, species are present. These issues are overcome by a double-dose kinetic analysis protocol. A rest state of [LACu(Et)(μ-TFA)(μ-{(enone)(ZnEt)2(enolate)})CuLA2]+ (through the equivalence of enolate = enone + ZnEt2) is supported by DFT studies (ωB97X-D/SRSC). Rate-determining ZnEt2(enone)2 transmetalation drives the exceptionally high catalytic activity of this system

Genetic Studies of Sulfadiazine-resistant and Methionine-requiring \u3cem\u3eNeisseria\u3c/em\u3e Isolated From Clinical Material

Deoxyribonucleate (DNA) preparations were extracted from Neisseria meningitidis (four isolates from spinal fluid and blood) and N. gonorrhoeae strains, all of which were resistant to sulfadiazine upon primary isolation. These DNA preparations, together with others from in vitro mutants of N. meningitidis and N. perflava, were examined in transformation tests by using as recipient a drug-susceptible strain of N. meningitidis (Ne 15 Sul-s Met+) which was able to grow in a methionine-free defined medium. The sulfadiazine resistance typical of each donor was introduced into the uniform constitution of this recipient. Production of p-aminobenzoic acid was not significantly altered thereby. Transformants elicited by DNA from the N. meningitidis clinical isolates were resistant to at least 200 μg of sulfadiazine/ml, and did not show a requirement for methionine (Sul-r Met+). DNA from six strains of N. gonorrhoeae, which were isolated during the period of therapeutic use of sulfonamides, conveyed lower degrees of resistance and, invariably, a concurrent methionine requirement (Sul-r/Met−). The requirement of these transformants, and that of in vitro mutants selected on sulfadiazine-agar, was satisfied by methionine, but not by vitamin B12, homocysteine, cystathionine, homoserine, or cysteine. Sul-r Met+ and Sul-r/Met− loci could coexist in the same genome, but were segregated during transformation. On the other hand, the dual Sul-r/Met− properties were not separated by recombination, but were eliminated together. DNA from various Sul-r/Met− clones tested against recipients having nonidentical Sul-r/Met− mutant sites yielded Sul-s Met+ transformants. The met locus involved is genetically complex, and will be a valuable tool for studies of genetic fine structure of members of Neisseria, and of genetic homology between species

Differential Single-stranded DNA Binding Properties of the Paralogous SsbA and SsbB Proteins from Streptococcus pneumoniae

The naturally transformable Gram-positive bacterium Streptococcus pneumoniae has two single-stranded DNA-binding (SSB) proteins, designated SsbA and SsbB. The SsbA protein is similar in size to the well characterized SSB protein from Escherichia coli (SsbEc). The SsbB protein, in contrast, is a smaller protein that is specifically induced during natural transformation and has no counterpart in E. coli. In this report, the single-stranded DNA (ssDNA) binding properties of the SsbA and SsbB proteins were examined and compared with those of the SsbEc protein. The ssDNA binding characteristics of the SsbA protein were similar to those of the SsbEc protein in every ssDNA binding assay used in this study. The SsbB protein differed from the SsbA and SsbEc proteins, however, both in its binding to short homopolymeric dT(n) oligomers (as judged by polyacrylamide gel-shift assays) and in its binding to the longer naturally occurring X and M13 ssDNAs (as judged by agarose gel-shift assays and electron microscopic analysis). The results indicate that an individual SsbB protein binds to ssDNA with an affinity that is similar or higher than that of the SsbA and SsbEc proteins. However, the manner in which multiple SsbB proteins assemble onto a ssDNA molecule differs from that observed with the SsbA and SsbEc proteins. These results represent the first analysis of paralogous SSB proteins from any bacterial species and provide a foundation for further investigations into the biological roles of these proteins

Doublet structures in quantum well absorption spectra due to Fano-related interference

In this theoretical investigation we predict an unusual interaction between a discrete state and a continuum of states, which is closely related to the case of Fano-interference. It occurs in a GaAs/AlxGa1-xAs quantum well between the lowest light-hole exciton and the continuum of the second heavy-hole exciton. Unlike the typical case for Fano-resonance, the discrete state here is outside the continuum; we use uniaxial stress to tune its position with respect to the onset of the continuum. State-of-the art calculations of absorption spectra show that as the discrete state approaches the continuum, a doublet structure forms which reveals anticrossing behaviour. The minimum separation energy of the anticrossing depends characteristically on the well width and is unusually large for narrow wells. This offers striking evidence for the strong underlying valence-band mixing. Moreover, it proves that previous explanations of similar doublets in experimental data, employing simple two-state models, are incomplete.Comment: 21 pages, 5 figures and 5 equations. Accepted for publication in Physical Review

Local radio to promote mental health awareness: a public health initiative.

BACKGROUND: Public health strategies have focused largely on physical health. However, there is increasing recognition that raising mental health awareness and tackling stigma is crucial to reduce disease burden. National campaigns have had some success but tackling issues locally is particularly important. AIMS: To assess the public's awareness and perception of the monthly BBC Cornwall mental health phone-in programmes that have run for 8.5 years in Cornwall, UK (population 530 000). METHOD: A consultation, review and feedback process involving a multiagency forum of mental and public health professionals, people with lived experience and local National Health Service trust's media team was used to develop a brief questionnaire. This was offered to all attendees at two local pharmacies covering populations of 27 000 over a 2-week period. RESULTS: In total, 14% (95% CI 11.9-16.5) were aware of the radio show, 11% (95% CI 9.0-13.1) have listened and the majority (76%) of those who listened did so more than once. The estimated reach is 70 000 people in the local population, of whom approximately 60 000 listen regularly. The show is highly valued among respondents with modal and median scores of 4 out of 5. CONCLUSIONS: Local radio is a successful, cost-effective and impactful way to reach a significant proportion of the population and likely to raise awareness, reduce stigma and be well received. The format has been adopted in other regions thus demonstrating easy transferability. It could form an essential part of a public health strategy to improve a population's mental well-being. DECLARATION OF INTEREST: W.H. received support from the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care (CLAHRC) for the South West Peninsula UK. The views expressed in this publication are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. L.R. and D.S. were involved in delivering the programmes but had no role in their evaluation

Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins

This study demonstrates mismatch repair (MMR) reactions reconstituted in vitro with purified Saccharomyces cerevisiae proteins. Biochemical analysis of MMR in vitro showed that MMR required mispair binding by the MutS homolog 2–MutS homolog 6 complex and corresponded to the Exonuclease 1-dependent subpathway of MMR. The reactions observed involved the formation of long excision tracts whose length was consistent with the length of MMR-dependent gene conversion tracts in vivo. The availability of this reconstituted MMR reaction now allows the wealth of mutations affecting MMR generated from the genetic analysis of S. cerevisiae MMR mechanisms in vivo to be used in biochemical reconstitution studies whose ultimate goal is to reconstitute MMR linked to both DNA replication and recombination