Intensive Mutagenesis of the Nisin Hinge Leads to the Rational Design of Enhanced Derivatives

peer-reviewedNisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid ‘hinge’ region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.This work was financed by a grant from the Irish Department of Agriculture, Food and the Marine through the Food Institutional Research Measure (08/RD/C/691) and with Science Foundation Investigator award (10/IN.1/B3027)

Assessing exposure of young children to common endocrine-disrupting chemicals in the home environment: A review and commentary of the questionnaire-based approach

Background: Although infants and young children are particularly vulnerable to endocrine disrupting chemical (EDC) exposure, there is an absence of comprehensive exposure data for this age group. As young children spend the majority of their time indoors, improved methods of exposure assessment are needed to characterise the health risks from exposures in the home environment. Biologic assessment, which has been considered the gold standard for exposure assessment in recent years, is difficult to conduct in young children. Questionnaires are an alternative and indirect method of predicting exposure, which may overcome some of the limitations of direct exposure assessment. Research problem: The feasibility of using a questionnaire-based approach to predict exposure of young children to EDCs in the home has yet to be comprehensively reviewed. Moreover, there is no one questionnaire that has been validated for predicting the exposure of infants to common EDCs in the home. Aims and objectives: The aim of this review is to discuss the use and validation of the questionnaire-based approach to predict exposure of children to chemicals from three common classes of EDCs in the home, namely, plasticisers, flame retardants, and insecticides. We discuss the strengths and weaknesses of the questionnaire-based approach as well as the important pathways of exposure in the home environment, by which to guide the design and validation of future exposure questionnaires. Results: The findings from our review indicate that the questionnaire-based approach is a valuable tool in the prediction of exposure to persistent organic pollutants, as well as to toxicants that have consistent patterns of exposure. With improvements to the design and validation process, the questionnaire-based approach may also prove to be a reliable instrument in predicting exposure to EDCs with short-half lives, including bisphenol A, phthalates, and pyrethroid and organophosphate insecticides

Porous silica spheres as indoor air pollutant scavengers

Porous silica spheres were investigated for their effectiveness in removing typical indoor air pollutants, such as aromatic and carbonyl-containing volatile organic compounds (VOCs), and compared to the commercially available polymer styrene-divinylbenzene (XAD-4). The silica spheres and the XAD-4 resin were coated on denuder sampling devices and their adsorption efficiencies for volatile organic compounds evaluated using an indoor air simulation chamber. Real indoor sampling was also undertaken to evaluate the affinity of the silica adsorbents for a variety of indoor VOCs. The silica sphere adsorbents were found to have a high affinity for polar carbonyls and found to be more efficient than the XAD-4 resin at adsorbing carbonyls in an indoor environment

Quantitative analysis of stylolite networks in different platform carbonate facies

Stylolites are rough surfaces that form by pressure solution, and present variable geometries and spatial distributions. Despite being ubiquitous in carbonate rocks and potentially influencing fluid flow, it is not yet clear how the type and distribution of stylolite networks relate to lithofacies. This study investigates Lower Cretaceous platform carbonates in the Benicàssim area (Maestrat Basin, Spain) to statistically characterise stylolite morphology and stylolite network distributions in a selection of typical shallow-marine carbonate lithofacies, from mudstones to grainstones. Bedding-parallel stylolite networks were sampled in the field to quantify stylolite spacing, wavelength, amplitude, intersection morphology and connectivity. Grain size, sorting and composition were found to be the key lithological variables responsible for the development of rough anastomosing stylolite networks. Poorly-connected stylolites with large vertical spacings were found to be dominant in grain-supported lithofacies, where grains are fine and well sorted. Anastomosing stylolite networks appear well developed in mud-supported lithofacies with poorly-sorted clasts that are both heterogenous in size and composition. Mud-supported facies feature stylolites that are closely spaced, have high amplitudes and intersection densities, and predominantly present suture and sharp-peak type morphologies. Larger grains and poor sorting favour the formation of stylolites with small vertical spacings, low wavelengths and high amplitudes. This statistical analysis approach requires only limited information, such as that from drill core, and can be used to characterise stylolite morphology and distributions in subsurface carbonate reservoirs

Chronic intermittent hypoxia increases rat sternohyoid muscle NADPH oxidase expression with attendant modest oxidative stress

Chronic intermittent hypoxia (CIH) causes upper airway muscle dysfunction. We hypothesized that the superoxide generating NADPH oxidase (NOX) is upregulated in CIH-exposed muscle causing oxidative stress. Adult male Wistar rats were exposed to intermittent hypoxia (5% O2 at the nadir for 90 s followed by 210 s of normoxia), for 8 h per day for 14 days. The effect of CIH exposure on the expression of NOX subunits, total myosin and 4-hydroxynonenal (4-HNE) protein adducts in sternohyoid muscle was determined by western blotting and densitometry. Sternohyoid protein free thiol and carbonyl group contents were determined by 1D electrophoresis using specific fluorophore probes. Aconitase and glutathione reductase activities were measured as indices of oxidative stress. HIF-1α content and key oxidative and glycolytic enzyme activities were determined. Contractile properties of sternohyoid muscle were determined ex vivo in the absence and presence of apocynin (putative NOX inhibitor). We observed an increase in NOX 2 and p47 phox expression in CIH-exposed sternohyoid muscle with decreased aconitase and glutathione reductase activities. There was no evidence, however, of increased lipid peroxidation or protein oxidation in CIH-exposed muscle. CIH exposure did not affect sternohyoid HIF-1α content or aldolase, lactate dehydrogenase, or glyceraldehyde-3-phosphate dehydrogenase activities. Citrate synthase activity was also unaffected by CIH exposure. Apocynin significantly increased sternohyoid force and power. We conclude that CIH exposure upregulates NOX expression in rat sternohyoid muscle with concomitant modest oxidative stress but it does not result in a HIF-1α-dependent increase in glycolytic enzyme activity. Constitutive NOX activity decreases sternohyoid force and power. Our results implicate NOX-dependent reactive oxygen species in CIH-induced upper airway muscle dysfunction which likely relates to redox modulation of key regulatory proteins in excitation-contraction coupling

Unrepeatered field transmission of 2 Tbit/s multi-banded coherent WDM over 124 km of installed SMF

In this paper we report field transmission of a 2Tbit/s multi-banded Coherent WDM signal over BT Ireland's installed SMF, using EDFA amplification only, with mixed Ethernet (with FEC) and PRBS payloads. To the best of our knowledge, the results obtained represent the highest total capacity transmitted over installed SMF with orthogonal subcarriers. BERs below 10(-5) and no frame-loss were recorded for all 49 subcarriers. Extended BER measurements over several hours showed fluctuations that can be attributed to PMD and to dynamic effects associated with clock instabilities

IL‐4 induces proliferation in prostate cancer PC3 cells under nutrient‐depletion stress through the activation of the JNK‐pathway and survivin up‐regulation

Interleukin (IL)‐4 plays a critical role in the regulation of immune responses and has been detected at high levels in the tumor microenvironment of cancer patients where it correlates with the grade of malignancy. The direct effect of IL‐4 on cancer cells has been associated with increased cell survival; however, its role in cancer cell proliferation and related mechanisms is still unclear. Here it was shown that in a nutrient‐depleted environment, IL‐4 induces proliferation in prostate cancer PC3 cells. In these cells, under nutrient‐depletion stress, IL‐4 activates mitogen‐activated protein kinases (MAPKs), including Erk, p38, and JNK. Using MAP‐signaling‐specific inhibitors, it was shown that IL‐4‐induced proliferation is mediated by JNK activation. In fact, JNK‐inhibitor‐V (JNKi‐V) stunted IL‐4‐mediated cell proliferation. Furthermore, it was found that IL‐4 induces survivin up‐regulation in nutrient‐depleted cancer cells. Using survivin‐short‐hairpin‐RNAs (shRNAs), it was demonstrated that in this milieu survivin expression above a threshold limit is critical to the mechanism of IL‐4‐mediated proliferation. In addition, the significance of survivin up‐regulation in a stressed environment was assessed in prostate cancer mouse xenografts. It was found that survivin knockdown decreases tumor progression in correlation with cancer cell proliferation. Furthermore, under nutrient depletion stress, IL ‐4 could induce proliferation in cancer cells from multiple origins: MDA‐MB‐231 (breast), A253 (head and neck), and SKOV‐3 (ovarian). Overall, these findings suggest that in a tumor microenvironment under stress conditions, IL‐4 triggers a simultaneous activation of the JNK‐pathway and the up‐regulation of survivin turning on a cancer proliferation mechanism. J. Cell. Biochem. 113: 1569–1580, 2012. © 2011 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90542/1/24025_ftp.pd

Experimental and modeled thermoregulatory costs of repeated sublethal oil exposure in the Double-crested Cormorant, \u3ci\u3ePhalacrocorax auritus\u3c/i\u3e

To fully understand the impact of oil exposure, it is important to understand sublethal effects like how increased thermoregulatory costs may affect survival and reproduction. However, it is difficult and time-consuming to measure these effects in wild animals. We present a novel use of a bioenergetics model, Niche Mapper™, to estimate thermoregulatory impacts of oiling, using data from captive Double-crested Cormorants (Phalacrocorax auritus) experimentally exposed to oil. Oiled cormorants had significant increases in surface body temperatures following exposure. Niche Mapper accurately predicted surface temperatures and metabolic rates for unoiled and oiled cormorants and predicted 13–18% increased daily energetic demands due to increased thermoregulatory costs of oiling, consistent with increased food consumption observed in experimentally oiled cormorants. We show that Niche Mapper can provide valuable insight into sublethal oiling effects by quantifying the extent to which thermoregulatory costs divert energy resources away from important life processes like maintenance, reproduction and migration

Polymorphism discovery and association analyses of the interferon genes in type 1 diabetes.

BACKGROUND: The aetiology of the autoimmune disease type 1 diabetes (T1D) involves many genetic and environmental factors. Evidence suggests that innate immune responses, including the action of interferons, may also play a role in the initiation and/or pathogenic process of autoimmunity. In the present report, we have adopted a linkage disequilibrium (LD) mapping approach to test for an association between T1D and three regions encompassing 13 interferon alpha (IFNA) genes, interferon omega-1 (IFNW1), interferon beta-1 (IFNB1), interferon gamma (IFNG) and the interferon consensus-sequence binding protein 1 (ICSBP1). RESULTS: We identified 238 variants, most, single nucleotide polymorphisms (SNPs), by sequencing IFNA, IFNB1, IFNW1 and ICSBP1, 98 of which where novel when compared to dbSNP build 124. We used polymorphisms identified in the SeattleSNP database for INFG. A set of tag SNPs was selected for each of the interferon and interferon-related genes to test for an association between T1D and this complex gene family. A total of 45 tag SNPs were selected and genotyped in a collection of 472 multiplex families. CONCLUSION: We have developed informative sets of SNPs for the interferon and interferon related genes. No statistical evidence of a major association between T1D and any of the interferon and interferon related genes tested was found.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Translations equations to compare ActiGraph GT3X and Actical accelerometers activity counts

Background: This study aimed to develop a translation equation to enable comparison between Actical and ActiGraph GT3X accelerometer counts recorded minute by minute. Methods: Five males and five females of variable height, weight, body mass index and age participated in this investigation. Participants simultaneously wore an Actical and an ActiGraph accelerometer for two days. Conversion algorithms and R2 were calculated day by day for each subject between the omnidirectional Actical and three different ActiGraph (three-dimensional) outputs: 1) vertical direction, 2) combined vector, and 3) a custom vector. Three conversion algorithms suitable for minute/minute conversions were then calculated from the full data set. Results: The vertical ActiGraph activity counts demonstrated the closest relationship with the Actical, with consistent moderate to strong conversions using the algorithm: y = 0.905x, in the day by day data (R2 range: 0.514 to 0.989 and average: 0.822) and full data set (R2 = 0.865). Conclusions: The Actical is most sensitive to accelerations in the vertical direction, and does not closely correlate with three-dimensional ActiGraph output. Minute by minute conversions between the Actical and ActiGraphvertical component can be confidently performed between data sets and might allow further synthesis of information between studies