SOME DYNAMIC MIXED BOUNDARY VALUE PROBLEMS IN LINEAR VISCOELASTICITY.

Dept. of Mathematics and Statistics. Paper copy at Leddy Library: Theses & Major Papers - Basement, West Bldg. / Call Number: Thesis1975 .S212. Source: Dissertation Abstracts International, Volume: 36-09, Section: B, page: 4534. Thesis (Ph.D.)--University of Windsor (Canada), 1975

The production of fluorescent transgenic trout to study in vitro myogenic cell differentiation

<p>Abstract</p> <p>Background</p> <p>Fish skeletal muscle growth involves the activation of a resident myogenic stem cell population, referred to as satellite cells, that can fuse with pre-existing muscle fibers or among themselves to generate a new fiber. In order to monitor the regulation of myogenic cell differentiation and fusion by various extrinsic factors, we generated transgenic trout (<it>Oncorhynchus mykiss</it>) carrying a construct containing the green fluorescent protein reporter gene driven by a fast myosin light chain 2 (MlC2f) promoter, and cultivated genetically modified myogenic cells derived from these fish.</p> <p>Results</p> <p>In transgenic trout, green fluorescence appeared in fast muscle fibers as early as the somitogenesis stage and persisted throughout life. Using an <it>in vitro </it>myogenesis system we observed that satellite cells isolated from the myotomal muscle of transgenic trout expressed GFP about 5 days post-plating as they started to fuse. GFP fluorescence persisted subsequently in myosatellite cell-derived myotubes. Using this <it>in vitro </it>myogenesis system, we showed that the rate of muscle cell differentiation was strongly dependent on temperature, one of the most important environmental factors in the muscle growth of poikilotherms.</p> <p>Conclusions</p> <p>We produced MLC2f-gfp transgenic trout that exhibited fluorescence in their fast muscle fibers. The culture of muscle cells extracted from these trout enabled the real-time monitoring of myogenic differentiation. This <it>in vitro </it>myogenesis system could have numerous applications in fish physiology to evaluate the myogenic activity of circulating growth factors, to test interfering RNA and to assess the myogenic potential of fish mesenchymal stem cells. In ecotoxicology, this system could be useful to assess the impact of environmental factors and marine pollutants on fish muscle growth.</p

Engineering of PA-IIL lectin from Pseudomonas aeruginosa – Unravelling the role of the specificity loop for sugar preference

<p>Abstract</p> <p>Background</p> <p>Lectins are proteins of non-immune origin capable of binding saccharide structures with high specificity and affinity. Considering the high encoding capacity of oligosaccharides, this makes lectins important for adhesion and recognition. The present study is devoted to the PA-IIL lectin from <it>Pseudomonas aeruginosa</it>, an opportunistic human pathogen capable of causing lethal complications in cystic fibrosis patients. The lectin may play an important role in the process of virulence, recognizing specific saccharide structures and subsequently allowing the bacteria to adhere to the host cells. It displays high values of affinity towards monosaccharides, especially fucose – a feature caused by unusual binding mode, where two calcium ions participate in the interaction with saccharide. Investigating and understanding the nature of lectin-saccharide interactions holds a great potential of use in the field of drug design, namely the targeting and delivery of active compounds to the proper site of action.</p> <p>Results</p> <p><it>In vitro </it>site-directed mutagenesis of the PA-IIL lectin yielded three single point mutants that were investigated both structurally (by X-ray crystallography) and functionally (by isothermal titration calorimetry). The mutated amino acids (22–23–24 triad) belong to the so-called specificity binding loop responsible for the monosaccharide specificity of the lectin. The mutation of the amino acids resulted in changes to the thermodynamic behaviour of the mutants and subsequently in their relative preference towards monosaccharides. Correlation of the measured data with X-ray structures provided the molecular basis for rationalizing the affinity changes. The mutations either prevent certain interactions to be formed or allow formation of new interactions – both of afore mentioned have strong effects on the saccharide preferences.</p> <p>Conclusion</p> <p>Mutagenesis of amino acids forming the specificity binding loop allowed identification of one amino acid that is crucial for definition of the lectin sugar preference. Altering specificity loop amino acids causes changes in saccharide-binding preferences of lectins derived from PA-IIL, via creation or blocking possible binding interactions. This finding opens a gate towards protein engineering and subsequent protein design to refine the desired binding properties and preferences, an approach that could have strong potential for drug design.</p

A qualitative assessment of the acceptability of hepatitis C remote self-testing and self-sampling amongst people who use drugs in London, UK.

BACKGROUND: Hepatitis C (HCV) diagnosis and care is a major challenge for people who use illicit drugs, and is characterised by low rates of testing and treatment engagement globally. New approaches to fostering engagement are needed. We explored the acceptability of remote forms of HCV testing including self-testing and self-sampling among people who use drugs in London, UK. METHODS: A qualitative rapid assessment was undertaken with people who use drugs and stakeholders in London, UK. Focus groups were held with men who have sex with men engaged in drug use, people who currently inject drugs and people who formerly injected drugs (22 participants across the 3 focus groups). Stakeholders participated in semi-structured interviews (n = 5). We used a thematic analysis to report significant themes in participants' responses. RESULTS: We report an overarching theme of 'tension' in how participants responded to the acceptability of remote testing. This tension is evident across four separate sub-themes we explore. First, choice and control, with some valuing the autonomy and privacy remote testing could support. Second, the ease of use of self testing linked to its immediate result and saliva sample was preferred over the delayed result from a self administered blood sample tested in a laboratory. Third, many respondents described the need to embed remote testing within a supportive care pathway. Fourth, were concerns over managing a positive result, and its different meanings, in isolation. CONCLUSIONS: The concept of remote HCV testing is acceptable to some people who use drugs in London, although tensions with lived experience of drug use and health system access limit its relevance. Future development of remote testing must respond to concerns raised in order for acceptable implementation to take place

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10