Cell Viability Assessment Using Fluorescence Vital Dyes and Confocal Microscopy in Evaluating Freezing and Thawing Protocols Used in Cryopreservation of Allogeneic Venous Grafts.

The authors present their contribution to the improvement of methods suitable for the detection of the freezing and thawing damage of cells of cryopreserved venous grafts used for lower limb revascularization procedures. They studied the post-thaw viability of cells of the wall of cryopreserved venous grafts (CVG) immediately after thawing and after 24 and 48 h culture at +37 °C in two groups of six CVG selected randomly for slow thawing in the refrigerator and rapid thawing in a water bath at +37 °C. The grafts were collected from multi-organ and tissue brain-dead donors, cryopreserved, and stored in a liquid nitrogen vapor phase for five years. The viability was assessed from tissue slices obtained by perpendicular and longitudinal cuts of the thawed graft samples using in situ staining with fluorescence vital dyes. The mean and median immediate post-thaw viability values above 70% were found in using both thawing protocols and both types of cutting. The statistically significant decline in viability after the 48-h culture was observed only when using the slow thawing protocol and perpendicular cutting. The possible explanation might be the "solution effect damage" during slow thawing, which caused a gentle reduction in the graft cellularity. The possible influence of this phenomenon on the immunogenicity of CVG should be the subject of further investigations.

Structural and Thermodynamic Analysis of the Resistance Development to Pimodivir (VX-787), the Clinical Inhibitor of Cap Binding to PB2 Subunit of Influenza A Polymerase.

Influenza A virus (IAV) encodes a polymerase composed of three subunits: PA, with endonuclease activity, PB1 with polymerase activity and PB2 with host RNA five-prime cap binding site. Their cooperation and stepwise activation include a process called cap-snatching, which is a crucial step in the IAV life cycle. Reproduction of IAV can be blocked by disrupting the interaction between the PB2 domain and the five-prime cap. An inhibitor of this interaction called pimodivir (VX-787) recently entered the third phase of clinical trial; however, several mutations in PB2 that cause resistance to pimodivir were observed. First major mutation, F404Y, causing resistance was identified during preclinical testing, next the mutation M431I was identified in patients during the second phase of clinical trials. The mutation H357N was identified during testing of IAV strains at Centers for Disease Control and Prevention. We set out to provide a structural and thermodynamic analysis of the interactions between cap-binding domain of PB2 wild-type and PB2 variants bearing these mutations and pimodivir. Here we present four crystal structures of PB2-WT, PB2-F404Y, PB2-M431I and PB2-H357N in complex with pimodivir. We have thermodynamically analysed all PB2 variants and proposed the effect of these mutations on thermodynamic parameters of these interactions and pimodivir resistance development. These data will contribute to understanding the effect of these missense mutations to the resistance development and help to design next generation inhibitors.

Impact assessment of pollutants from waste-related operations as a feature of holistic logistic tool.

Waste management has still been a developing and progressing field, which demands continual improvements in waste transportation as well as proper selection of locations and technical operation of new treatment facilities. Most of research papers on waste management planning have been dealing with optimisation of network flows, thus minimising the cost and improving economic criteria. The shortest paths to treatment facilities are considered together with detailed analysis of their operation including heat and electricity demands in their vicinity. The tasks sometimes include social and global environmental criterions, however, the direct local consequences also play an important role and should be examined. A decision-making strategy in waste management updated with the local emission impact on the population is proposed in this paper. The paper focuses on the first move in analysing the production, dispersion, and impact of pollutants, originating in transport, with regards to the population living close to routes. The calculation of emission produced during the transport of waste takes into consideration the altitude profiles of routes, container loads, and specific types of vehicles. The consecutive estimated impact on the population reckons with the distances between routes and municipalities as well as their sizes in terms of the numbers of inhabitants, where the transportation routes are divided into smaller segments and dispersion is limited with threshold value. The proposed approach describing the emission effect has been tested using real-life operating data corresponding to the specific, 81 km long route along which approximately 25 t of waste is transported 800 times a year. The impact of pollutants on the population was evaluated and discussed. Results of the analysis were quantified for this route to create an edge characterisation needed for further calculations. This approach applied to the whole network then yields input data needed for future research of novel strategies in facility location problems. Other possible extensions of the presented approach include more accurate dispersion function or detailed calculation of the impact of pollutants with respect to specific locations of residential houses.