Rapid identification of precursor cDNAs encoding five structural classes of antimicrobial peptides from pickerel frog (Rana palustris) skin secretion by single step “shotgun” cloning.

The skin secretion of the North American pickerel frog (Rana palustris) has long been known to have pronounced noxious/toxic properties and to be highly effective in defence against predators and against other sympatric amphibians. As it consists largely of a complex mixture of peptides, it has been subjected to systematic peptidomic study but there has been little focus on molecular cloning of peptide-encoding cDNAs and by deduction, the biosynthetic precursors that they encode. Here, we demonstrate that the cDNAs encoding the five major structural families of antimicrobial peptides can be elucidated by a single step “shotgun” cloning approach using a cDNA library constructed from the source material of the peptidomic studies—the defensive skin secretion itself. Using a degenerate primer pool designed to a highly conserved nucleic acid sequence 5' to the initiation codon of known antimicrobial peptide precursor transcripts, we amplified cDNA sequences representing five major classes of antimicrobial peptides, such as esculentins, brevinins, ranatuerins, palustrins and temporins. Bioinformatic comparisons of precursor open-reading frames and nucleic acid sequences revealed high degrees of structural similarities between analogous peptides of R. palustris and the Chinese bamboo odorous frog, Rana versabilis. This approach thus constitutes a robust technique that can be used either alone or ideally, in parallel with peptidomic analysis of skin secretion, to rapidly extract primary structural information on amphibian skin secretion peptides and their biosynthetic precursors.

Rapid differentiation between fluconazole-sensitive and -resistant species of Candida directly from positive blood-culture bottles by real-time PCR

In view of both the delay in obtaining identification by conventional methods following blood-culture positivity in patients with candidaemia and the close relationship between species and fluconazole (FLC) susceptibility, early speciation of positive blood cultures has the potential to influence therapeutic decisions. The aim was to develop a rapid test to differentiate FLC-resistant from FLC-sensitive Candida species. Three TaqMan-based real-time PCR assays were developed to identify up to six Candida species directly from BacT/Alert blood-culture bottles that showed yeast cells on Gram staining at the time of initial positivity. Target sequences in the rRNA gene complex were amplified, using a consensus two-step PCR protocol, to identify Candida albicans, Candida parapsilosis, Candida tropicalis, Candida dubliniensis, Candida glabrata and Candida krusei; these are the most commonly encountered Candida species in blood cultures. The first four of these (the characteristically FLC-sensitive group) were identified in a single reaction tube using one fluorescent TaqMan probe targeting 1 8S rRNA sequences conserved in the four species. The FLC-resistant species C. krusei and C. glabrata were detected in two further reactions, each with species-specific probes. This method was validated with clinical specimens (blood cultures) positive for yeast (n=33 sets) and the results were 100% concordant with those of phenotypic identification carried out concomitantly. The reported assay significantly reduces the time required to identify the presence of C. glabrata and C. krusei in comparison with a conventional phenotypic method, from ~72 to

One-step synthesis of ZnO nanosheets: a blue-white fluorophore

Zinc oxide is synthesised at low temperature (80A degrees C) in nanosheet geometry using a substrate-free, single-step, wet-chemical method and is found to act as a blue-white fluorophore. Investigation by atomic force microscopy, electron microscopy, and X-ray diffraction confirms zinc oxide material of nanosheet morphology where the individual nanosheets are polycrystalline in nature with the crystalline structure being of wurtzite character. Raman spectroscopy indicates the presence of various defects, while photoluminescence measurements show intense green (centre wavelength approximately 515 nm) blue (approximately 450 nm), and less dominant red (approximately 640 nm) emissions due to a variety of vacancy and interstitial defects, mostly associated with surfaces or grain boundaries. The resulting colour coordinate on the CIE-1931 standard is (0.23, 0.33), demonstrating potential for use as a blue-white fluorescent coating in conjunction with ultraviolet emitting LEDs. Although the defects are often treated as draw-backs of ZnO, here we demonstrate useful broadband visible fluorescence properties in as-prepared ZnO. 

Rational Attenuation of a Morbillivirus by Modulating the Activity of the RNA-Dependent RNA Polymerase

Negative-strand RNA viruses encode a single RNA-dependent RNA polymerase (RdRp) which transcribes and replicates the genome. The open reading frame encoding the RdRp from a virulent wild-type strain of rinderpest virus (RPV) was inserted into an expression plasmid. Sequences encoding enhanced green fluorescent protein (EGFP) were inserted into a variable hinge of the RdRp. The resulting polymerase was autofluorescent, and its activity in the replication/transcription of a synthetic minigenome was reduced. We investigated the potential of using this approach to rationally attenuate a virus by inserting the DNA sequences encoding the modified RdRp into a full-length anti-genome plasmid from which a virulent virus (rRPV(KO)) can be rescued. A recombinant virus, rRPV(KO)L-RRegfpR, which grew at an indistinguishable rate and to an identical titer as rRPV(KO) in vitro, was rescued. Fluorescently tagged polymerase was visible in large cytoplasmic inclusions and beneath the cell membrane. Subcutaneous injection of 10(4) TCID(50) of the rRPV(KO) parental recombinant virus into cattle leads to severe disease symptoms (leukopenia/diarrhea and pyrexia) and death by 9 days postinfection. Animals infected with rRPV(KO)L-RRegfpR exhibited transient leukopenia and mild pyrexia, and the only noticeable clinical signs were moderate reddening of one eye and a slight ocular-nasal discharge. Viruses that expressed the modified polymerase were isolated from peripheral blood lymphocytes and eye swabs. This demonstrates that a virulent morbillivirus can be attenuated in a single step solely by modulating RdRp activity and that there is not necessarily a correlation between virus growth in vitro and in vivo.

Risk assessment of E-Commerce projects using evidential reasoning

The purpose of this study is to develop a decision making system to evaluate the risks in E-Commerce (EC) projects. Competitive software businesses have the critical task of assessing the risk in the software system development life cycle. This can be conducted on the basis of conventional probabilities, but limited appropriate information is available and so a complete set of probabilities is not available. In such problems, where the analysis is highly subjective and related to vague, incomplete, uncertain or inexact information, the Dempster-Shafer (DS) theory of evidence offers a potential advantage. We use a direct way of reasoning in a single step (i.e., extended DS theory) to develop a decision making system to evaluate the risk in EC projects. This consists of five stages 1) establishing knowledge base and setting rule strengths, 2) collecting evidence and data, 3) determining evidence and rule strength to a mass distribution for each rule; i.e., the first half of a single step reasoning process, 4) combining prior mass and different rules; i.e., the second half of the single step reasoning process, 5) finally, evaluating the belief interval for the best support decision of EC project. We test the system by using potential risk factors associated with EC development and the results indicate that the system is promising way of assisting an EC project manager in identifying potential risk factors and the corresponding project risks.

Elimination kinetic of 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate ester metabolites in calves' urine

Efficient control of the illegal use of anabolic steroids must both take into account metabolic patterns and associated kinetics of elimination; in this context, an extensive animal experiment involving 24 calves and consisting of three administrations of 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate esters was carried out over 50 days. Urine samples were regularly collected during the experiment from all treated and non-treated calves. For sample preparation, a single step high throughput protocol based on 96-well C-18 SPE was developed and validated according to the European Decision 2002/657/EC requirements. Decision limits (CC alpha) for steroids were below 0.1 mu g L-1, except for 19-norandrosterone (CC alpha = 0.7 mu g L-1) and estrone (CC alpha = 0.3 mu g L-1). Kinetics of elimination of the administered 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate were established by monitoring 17 beta-estradiol, 17 alpha-estradiol, estrone and 17 beta-nandrolone, 17 alpha-nandrolone, 19-noretiocholanolone, 19-norandrostenedione, respectively. All animals demonstrated homogeneous patterns of elimination both from a qualitative (metabolite profile) and quantitative point of view (elimination kinetics in urine). Most abundant metabolites were 17 alpha-estradiol and 17 alpha-nandrolone (> 20 and 2 mg L-1, respectively after 17 beta-estradiol 3-benzoate and 17 beta-nandrolone laureate administration) whereas 17 beta-estradiol, estrone, 17 beta-nandrolone, 19-noretiocholanolone and 19-norandrostenedione were found as secondary metabolites at concentration values up to the mu g L-1 level. No significant difference was observed between male and female animals. The effect of several consecutive injections on elimination profiles was studied and revealed a tendency toward a decrease in the biotransformation of administered steroid 17 beta form. (c) 2008 Elsevier Ltd. All rights reserved.

Laser-engineered dissolving microneedles for active transdermal delivery of nadroparin calcium

There is an urgent need to replace the injection currently used for low molecular weight heparin (LMWH) multidose therapy with a non- or minimally invasive delivery approach. In this study, laser-engineered dissolving microneedle (DMN) arrays fabricated from aqueous blends of 15% w/w poly(methylvinylether-co-maleic anhydride) were used for the first time in active transdermal delivery of the LMWH nadroparin calcium (NC). Importantly, an array loading of 630 IU of NC was achieved without compromising the array mechanical strength or drug bioactivity. Application of NC-DMNs to dermatomed human skin (DHS) using the single-step 'poke and release' approach allowed permeation of approximately 10.6% of the total NC load over a 48-h study period. The cumulative amount of NC that permeated DHS at 24 h and 48 h attained 12.28 ± 4.23 IU/cm and 164.84 ± 8.47 IU/cm , respectively. Skin permeation of NC could be modulated by controlling the DMN array variables, such as MN length and array density as well as application force to meet various clinical requirements including adjustment for body mass and renal function. NC-loaded DMN offers great potential as a relatively low-cost functional delivery system for enhanced transdermal delivery of LMWH and other macromolecules. © 2012 Elsevier B.V. All rights reserved.

Mechanochemical interconversion between discrete complexes and coordination networks - formal hydration/dehydration by LAG

Using a small planetary ball mill, liquid-assisted grinding (LAG) of metal salts or oxides (ZnO, CdO, CdCO3, Cu(OAc)(2)center dot H2O, Co(OAc)(2)center dot 4H(2)O, Mn(OAc)(2)center dot 4H(2)O, Ni(OAc)(2)center dot 4H(2)O, FeSO4 center dot 7H(2)O) with two equivalents of isonicotinic acid (HINA) and small amounts of water ( up to 5.6 molar equivalents) gave discrete aquo complexes trans-[M(INA)(2)(OH2)(4)] (M = Zn, Cd, Cu, Fe, Co, Ni, Mn) efficiently within 30 min. For M = Zn, Cd and Cu these complexes readily undergo reversible formal dehydration to the extended network structures [M(INA)(2)] (M = Zn, Cu) or [Cd(INA)(2)(OH2)]center dot DMF by further LAG with non-aqueous liquids such as methanol or DMF. Overall, the mechanochemical dehydrations are more effective than heating or immersion in bulk solvents. The work demonstrates a two-step mechanochemical synthesis of coordination networks via discrete aquo complexes which may be preferable to single step reactions or grinding-annealing procedures in some cases. For example, the two step method was the only way to prepare [Cd(INA)(2)(OH2)]center dot DMF mechanochemically and the porous network Cu(INA)(2) could not be obtained from the aquo complex by heating.

Development and Validation of a Novel Lateral Flow Immunoassay (LFIA) for the Rapid Screening of Paralytic Shellfish Toxins (PSTs) from Shellfish Extracts

A single-step lateral flow immunoassay (LFIA) was developed and validated for the rapid screening of paralytic shellfish toxins (PSTs) from a variety of shellfish species, at concentrations relevant to regulatory limits of 800 μg STX-diHCl equivalents/kg shellfish meat. A simple aqueous extraction protocol was performed within several minutes from sample homogenate. The qualitative result was generated after a 5 min run time using a portable reader which removed subjectivity from data interpretation. The test was designed to generate noncompliant results with samples containing approximately 800 μg of STX-diHCl/kg. The cross-reactivities in relation to STX, expressed as mean ± SD, were as follows: NEO: 128.9% ± 29%; GTX1&4: 5.7% ± 1.5%; GTX2&3: 23.4% ± 10.4%; dcSTX: 55.6% ± 10.9%; dcNEO: 28.0% ± 8.9%; dcGTX2&3: 8.3% ± 2.7%; C1&C2: 3.1% ± 1.2%; GTX5: 23.3% ± 14.4% (n = 5 LFIA lots). There were no indications of matrix effects from the different samples evaluated (mussels, scallops, oysters, clams, cockles) nor interference from other shellfish toxins (domoic acid, okadaic acid group). Naturally contaminated sample evaluations showed no false negative results were generated from a variety of different samples and profiles (n = 23), in comparison to reference methods (MBA method 959.08, LC-FD method 2005.06). External laboratory evaluations of naturally contaminated samples (n = 39) indicated good correlation with reference methods (MBA, LC-FD). This is the first LFIA which has been shown, through rigorous validation, to have the ability to detect most major PSTs in a reliable manner and will be a huge benefit to both industry and regulators, who need to perform rapid and reliable testing to ensure shellfish are safe to eat.

Prevalence of the prothrombin G20210A mutation in the Irish populations:use of a novel polymerase chain reaction approach

The prothrombin G20210A polymorphism is associated with a threefold-increased risk of venous thrombosis. There is considerable variation in the reported prevalence of this polymorphism within normal populations, ranging from 0 to 6.5%. The prevalence within the Irish population has not been determined. A restriction fragment length polymorphism (RFLP)-based assay is commonly used for the detection of the prothrombin 20210A allele. This assay does not include a control restriction digest fragment and, consequently, failure of the enzyme activity or lack of addition of enzyme to the sample cannot be distinguished from wild-type prothrombin. We developed a RFLP-based assay, which incorporates an invariant digest site, resulting in the generation of a control digest fragment. Furthermore, we developed a nested polymerase chain reaction (PCR) method for the amplification and digestion of poor-quality or low-concentration DNA. In the Irish population studied, five of 385 (1.29%) were heterozygous and one patient was homozygous for the prothrombin 20210A polymorphism. This is the first reported data on an Irish or Celtic population and suggests that the allele frequency is similar to Anglo-Saxon populations. The nested PCR method successfully amplified and digested 100/100 (100%) of the archived samples; none of these samples could be analyzed by the standard single-round PCR method. In conclusion, nested PCR should be considered in the analysis of archived samples. Single-round PCR is appropriate for recently collected samples; however, an invariant control digest site should be incorporated in RFLP-based assays to validate the integrity of the digestion enzyme and limit the risk of false-negative results.

Development and Validation of a Lateral Flow Immunoassay for the Rapid Screening of Okadaic Acid and All Dinophysis Toxins from Shellfish Extracts

A single-step lateral flow immunoassay was developed and validated to detect okadaic acid (OA) and dinophysis toxins (DTXs), which cause diarrhetic shellfish poisoning. The performance characteristics of the test were investigated, in comparison to reference methods (liquid chromatography tandem mass spectrometry and/or bioassay), using both spiked and naturally contaminated shellfish. A portable reader was used to generate a qualitative result, indicating the absence or presence of OA-group toxins, at concentrations relevant to the maximum permitted level (MPL). Sample homogenates could be screened in 20 min (including extraction and assay time) for the presence of free toxins (OA, DTX1, DTX2). DTX3 detection could be included with the addition of a hydrolysis procedure. No matrix effects were observed from the species evaluated (mussels, scallops, oysters, and clams). Results from naturally contaminated samples (n = 72) indicated no false compliant results and no false noncompliant results at <50% MPL. Thus, the development of a new low-cost but highly effective tool for monitoring a range of important phycotoxins has been demonstrated.

An updated comprehensive techno-economic analysis of algae biodiesel

Algae biodiesel is a promising but expensive alternative fuel to petro-diesel. To overcome cost barriers, detailed cost analyses are needed. A decade-old cost analysis by the U.S. National Renewable Energy Laboratory indicated that the costs of algae biodiesel were in the range of $0.53–0.85/L (2012 USD values). However, the cost of land and transesterification were just roughly estimated. In this study, an updated comprehensive techno-economic analysis was conducted with optimized processes and improved cost estimations. Latest process improvement, quotes from vendors, government databases, and other relevant data sources were used to calculate the updated algal biodiesel costs, and the final costs of biodiesel are in the range of $0.42–0.97/L. Additional improvements on cost-effective biodiesel production around the globe to cultivate algae was also recommended. Overall, the calculated costs seem promising, suggesting that a single step biodiesel production process is close to commercial reality.

Automated Equation Formulation for Causal Loop Diagrams

The annotation of Business Dynamics models with parameters and equations, to simulate the system under study and further evaluate its simulation output, typically involves a lot of manual work. In this paper we present an approach for automated equation formulation of a given Causal Loop Diagram (CLD) and a set of associated time series with the help of neural network evolution (NEvo). NEvo enables the automated retrieval of surrogate equations for each quantity in the given CLD, hence it produces a fully annotated CLD that can be used for later simulations to predict future KPI development. In the end of the paper, we provide a detailed evaluation of NEvo on a business use-case to demonstrate its single step prediction capabilities.