99 research outputs found

    Circular Permutation in the Ω-Loop of TEM-1 β-Lactamase Results in Improved Activity and Altered Substrate Specificity

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    Generating diverse protein libraries that contain improved variants at a sufficiently high frequency is critical for improving the properties of proteins using directed evolution. Many studies have illustrated how random mutagenesis, cassette mutagenesis, DNA shuffling and similar approaches are effective diversity generating methods for directed evolution. Very few studies have explored random circular permutation, the intramolecular relocation of the N- and C-termini of a protein, as a diversity-generating step for directed evolution. We subjected a library of random circular permutations of TEM-1 β-lactamase to selections on increasing concentrations of a variety of β-lactam antibiotics including cefotaxime. We identified two circularly permuted variants that conferred elevated resistance to cefotaxime but decreased resistance to other antibiotics. These variants were circularly permuted in the Ω-loop proximal to the active site. Remarkably, one variant was circularly permuted such that the key catalytic residue Glu166 was located at the N-terminus of the mature protein

    Tumor-derived GDF-15 blocks LFA-1 dependent T cell recruitment and suppresses responses to anti-PD-1 treatment

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    Immune checkpoint blockade therapy is beneficial and even curative for some cancer patients. However, the majority don’t respond to immune therapy. Across different tumor types, pre-existing T cell infiltrates predict response to checkpoint-based immunotherapy. Based on in vitro pharmacological studies, mouse models and analyses of human melanoma patients, we show that the cytokine GDF-15 impairs LFA-1/β2-integrin-mediated adhesion of T cells to activated endothelial cells, which is a pre-requisite of T cell extravasation. In melanoma patients, GDF-15 serum levels strongly correlate with failure of PD-1-based immune checkpoint blockade therapy. Neutralization of GDF-15 improves both T cell trafficking and therapy efficiency in murine tumor models. Thus GDF-15, beside its known role in cancer-related anorexia and cachexia, emerges as a regulator of T cell extravasation into the tumor microenvironment, which provides an even stronger rationale for therapeutic anti-GDF-15 antibody development. Experimental cancer immunology and therap

    Arthropod venom Hyaluronidases: biochemical properties and potential applications in medicine and biotechnology

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    Ethical challenges in nursing homes - staff's opinions and experiences with systematic ethics meetings with participation of residents' relatives.

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    Background Many ethical problems exist in nursing homes. These include, for example, decision-making in end-of-life care, use of restraints and a lack of resources. Aims The aim of the present study was to investigate nursing home staffs’ opinions and experiences with ethical challenges and to find out which types of ethical challenges and dilemmas occur and are being discussed in nursing homes. Methods The study used a two-tiered approach, using a questionnaire on ethical challenges and systematic ethics work, given to all employees of a Norwegian nursing home including nonmedical personnel, and a registration of systematic ethics discussions from an Austrian model of good clinical practice. Results Ninety-one per cent of the nursing home staff described ethical problems as a burden. Ninety per cent experienced ethical problems in their daily work. The top three ethical challenges reported by the nursing home staff were as follows: lack of resources (79%), end-of-life issues (39%) and coercion (33%). To improve systematic ethics work, most employees suggested ethics education (86%) and time for ethics discussion (82%). Of 33 documented ethics meetings from Austria during a 1-year period, 29 were prospective resident ethics meetings where decisions for a resident had to be made. Agreement about a solution was reached in all 29 cases, and this consensus was put into practice in all cases. Residents did not participate in the meetings, while relatives participated in a majority of case discussions. In many cases, the main topic was end-of-life care and life-prolonging treatment. Conclusions Lack of resources, end-of-life issues and coercion were ethical challenges most often reported by nursing home staff. The staff would appreciate systematic ethics work to aid decision-making. Resident ethics meetings can help to reach consensus in decision-making for nursing home patients. In the future, residents’ participation should be encouraged whenever possible
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