Development of a multiplex flow cytometric microsphere immunoassay for mycotoxins and evaluation of its application in feed

A multi-mycotoxin immunoassay—using the MultiAnalyte Profiling (xMAP) technology—is developed and evaluated. This technology combines a unique color-coded microsphere suspension array, with a dedicated flow cytometer. We aimed for the combined detection of aflatoxins, ochratoxin A, deoxynivalenol, fumonisins, zearalenone and T-2-toxin in an inhibition immunoassay format. Sets of six mycotoxin-protein conjugates and six specific monoclonal antibodies were selected, and we observed good sensitivities and no cross-interactions between the assays in buffer. However, detrimental effects of the feed extract on the sensitivities and in some cases on the slopes of the curves were observed and different sample materials showed different effects. Therefore, for quantitative analysis, this assay depends on calibration curves in blank matrix extracts or on the use of a suitable multi-mycotoxin cleanup. To test if the method was suitable for the qualitative detection at EU guidance levels, we fortified rapeseed meal, a feed ingredient, with the six mycotoxins, and all extracts showed inhibited responses in comparison with the non-fortified sample extract. Contaminated FAPAS reference feed samples assigned for a single mycotoxin showed strong inhibitions in the corresponding assays but also often in other assays of the multiplex. In most cases, the presence of these other mycotoxins was confirmed by instrumental analysis. The multiplex immunoassay can be easily extended with other mycotoxins of interest, but finding a suitable multi-mycotoxin cleanup will improve its applicability

The appeal of the Functional Fitness MOT to older adults and health professionals in an outpatient setting: a mixed-method feasibility study

Purpose: To understand the views and perceptions regarding the Functional Fitness MOT (FFMOT), a battery of functional tests followed by a brief motivational interview, of both the older people undergoing it and the health professionals delivering it. Patients and methods: Physically inactive older adults (n=29) underwent the FFMOT and subsequently attended focus groups to share their perceptions of it and to discuss the barriers, motivators, health behavior change, and scope to improve physical activity (PA) levels. PA levels were recorded at baseline and again at 12 weeks together with a post-intervention questionnaire concerning behavior change. Participating physiotherapists and technical instructors were interviewed. Results: Most participants felt they had learned about their abilities and comparisons with their peers, had a change in perception about the importance of good balance and strength, and felt the FFMOT helped raise their awareness of local and self-directed physical activity opportunities. Most felt their awareness of the need for PA had not changed, but 25% of participants started a new organized PA opportunity. The health professionals perceived the FFMOT as being easy to administer, educating, and motivating for participants to increase their PA. Space, time, finances, and insecurity about having the necessary skills to conduct the FFMOTs were seen as barriers in implementing the FFMOT in daily practice. Conclusion: Over half of those offered the FFMOT accepted it, suggesting it is appealing. However, most participants felt they were already active enough and that their awareness of the need for PA had not changed. There were positive perceptions of the FFMOT from both professionals and older people, but both felt the FFMOT could be held in a community venue. The overall findings suggest that the FFMOT is feasible in the clinical setting, but its effectiveness has yet to be determined

Protein Design Based on a PHD Scaffold

The plant homeodomain (PHD) is a protein domain of ~45�100 residues characterised by a Cys4-His-Cys3 zinc-binding motif. When we commenced our study of the PHD in 2000, it was clear that the domain was commonly found in proteins involved in transcription. Sequence alignments indicate that while the cysteines, histidine and a few other key residues are strictly conserved, the rest of the domain varies greatly in terms of both amino acid composition and length. However, no structural information was available on the PHD and little was known about its function. We were therefore interested in determining the structure of a PHD in the hope that this might shed some light on its function and molecular mechanism of action. Our work began with the structure determination of a representative PHD, Mi2b-P2, and this work is presented in Chapter 3. Through comparison of this structure with the two other PHD structures that were determined during the course of our work, it became clear that PHDs adopt a well-defined globular fold with a superimposable core region. In addition, PHDs contain two loop regions (termed L1 and L3) that display increased flexibility and overlay less well between the three PHD structures available. These L1 and L3 regions correspond to variable regions identified earlier in PHD sequence alignments, indicating that L1 and L3 are probably not crucial for the PHD fold, but are instead likely to be responsible for imparting function(s) to the PHD. Indeed, numerous recent functional studies of PHDs from different proteins have since demonstrated their ability in binding a range of other proteins. In order to ascertain whether or not L1 and L3 were in fact dispensable for folding, we made extensive mutations (including both insertions and substitutions) in the loop regions of Mi2b-P2 and showed that the structure was maintained. We then went on to illustrate that a new function could be imparted to Mi2b-P2 by inserting a five-residue CtBP-binding motif into the L1 region and showed this chimera could fold and bind CtBP. Having established that the PHD could adopt a new binding function, we next sought to use combinatorial methods to introduce other novel functions into the PHD scaffold. Phage display was selected for this purpose, because it is a well-established technique and has been used successfully to engineer zinc-binding domains by other researchers. However, in order to establish this technique in our laboratory, we first chose a control system in which two partner proteins were already known to interact in vitro. We chose the protein complex formed between the transcriptional regulators LMO2 and ldb1 as a test case. We have examined this interaction in detail in our laboratory, and determined its three-dimensional structure. Furthermore, inappropriate formation of this complex is implicated in the onset of T-cell acute lymphoblastic leukemia. We therefore sought to use phage display to engineer ldb1 mimics that could potentially compete against wild-type ldb1 for LMO2, and this work is described in Chapter 4. Using a phage library containing ~3 x 10 7 variants of the LMO2-binding region of ldb1, we isolated mutants that were able to interact with LMO2 with higher affinity and specificity than wild-type ldb1. These ldb1 mutants represent a first step towards finding potential therapeutics for treating LMO-associated diseases. Having established phage display in our laboratory, we went on to search for PHD mutants that could bind selected target proteins. This work is described in Chapter 5. We created three PHD libraries with eight randomized residues in each of L1, L3 or in both loops of the PHD. These PHD libraries were then screened against four target proteins. After four rounds of selection, we were able to isolate a PHD mutant (dubbed L13-FH6) that could bind our test protein Fli-ets. This result demonstrates that a novel function can be imparted to the PHD using combinatorial methods and opens the way for further work in applying the PHD scaffold to other protein design work. In summary, the work detailed in Chapters 3 and 5 demonstrates that the PHD possesses many of the properties that are desirable for a protein scaffold for molecular recognition, including small size, stability, and a well-characterised structure. Moreover, the PHD motif possesses two loops (L1 and L3) of substantial size that can be remodeled for target binding. This may lead to an enhancement of binding affinities and specificities over other small scaffolds that have only one variable loop. In light of the fact that PHDs are mainly found in nuclear proteins, it is reasonable to expect that engineered PHDs could be expressed and function in an intracellular environment, unlike many other scaffolds that can only function in an oxidizing environment. Therefore, our results together with other currently available genomic and functional information indicate PHD is an excellent candidate for a scaffold that could be used to modify cellular processes. Appendices 1 and 2 describe completed bodies of work on unrelated projects that I have carried out during the course of my PhD candidature. The first comprises the invention and application of DNA sequences that contain all N-base sequences in the minimum possible length. This work is presented as a reprint of our recently published paper in Nucleic Acids Research. The second Appendix describes our structural analysis of an antifreeze protein from the shorthorn sculpin, a fish that lives in the Arctic and Antarctic oceans. This work is presented as a manuscript that is currently under review at the Journal of the American Chemical Society

Centaur 1952

Digitised by the Faculty of the Veterinary Scienc

Lost in translation? Standardising the terminology used in marine invasion biology and updating South African alien species lists

Confusion between terms and ambiguities among definitions have long plagued the field of invasion biology. One result is disruption in flow of information from researchers to policy-makers and managers who rely on science to inform regulatory frameworks and management actions. We reviewed the South African marine biology literature to quantify the current usage of terminology describing marine invasions and found a variety of terms in use, few of which are defined when used. In response, we propose standard terminology that aligns with international practice. We then interpreted the Blackburn unified framework for biological invasions within the marine context and used this as a transparent way to apply the standardised terms to an updated list of marine alien species for the country. This resulted in the recognition of 36 alien and 53 invasive species within South Africa. Most notably, follow-up research is required to confirm the status of at least 11 listed species, the majority of which have been recorded only once, or not in the past 25 years. It is hoped that by standardising terminology, marine science in South Africa will better support authorities charged with managing the threat posed by marine alien species

BEW: Bioinformatics workbench for analysis of biofilms experimental data

Biofilms research has evolved considerably in the last decade and is now generating large volumes of heterogeneous data. MIABiE, the international initiative on Biofilms, is devising guidelines for data interchange, and some databases provide access to biofilms experiments. However, the field is lacking appropriate bioinformatics tools in support of increasing operational and analytical needs. This paper presents a flexible and extensible open-source workbench for the operation and analysis of biofilms experiments, as follows: (i) the creation of customised experiments, (ii) the collection of various analytical results, following community standardisation guidelines and (iii) on-demand reporting and statistical evaluation

Arecibo-green bank-LOFAR carbon radio recombination line observations toward cold H I clouds

Interstellar matter and star formatio

On the Circular Orbit Approximation for Binary Compact Objects In General Relativity

One often-used approximation in the study of binary compact objects (i.e., black holes and neutron stars) in general relativity is the instantaneously circular orbit assumption. This approximation has been used extensively, from the calculation of innermost circular orbits to the construction of initial data for numerical relativity calculations. While this assumption is inconsistent with generic general relativistic astrophysical inspiral phenomena where the dissipative effects of gravitational radiation cause the separation of the compact objects to decrease in time, it is usually argued that the timescale of this dissipation is much longer than the orbital timescale so that the approximation of circular orbits is valid. Here, we quantitatively analyze this approximation using a post-Newtonian approach that includes terms up to order ({Gm/(rc^2)})^{9/2} for non-spinning particles. By calculating the evolution of equal mass black hole / black hole binary systems starting with circular orbit configurations and comparing them to the more astrophysically relevant quasicircular solutions, we show that a minimum initial separation corresponding to at least 6 (3.5) orbits before plunge is required in order to bound the detection event loss rate in gravitational wave detectors to < 5% (20%). In addition, we show that the detection event loss rate is > 95% for a range of initial separations that include all modern calculations of the innermost circular orbit (ICO).Comment: 10 pages, 12 figures, revtex