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Despite the high prevalence of BK polyomavirus (BKPyV) and the associated risk for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant (KTX) recipients, many details on viral processes such as replication, maturation, assembly and virion release from host cells have not been fully elucidated. VP1 is a polyomavirus-specific protein that is expressed in the late phase of its replicative cycle with important functions in virion assembly and infectious particle release. This study investigated the localization and time-dependent changes in the distribution of VP1-positive viral particles and their association within the spectrum of differing cell morphologies that are observed in the urine of KTX patients upon active BKPyV infection. We found highly differing recognition patterns of two anti-VP1 antibodies with respect to intracellular and extracellular VP1 localization, pointing towards independent binding sites that were seemingly associated with differing stages of virion maturation. Cells originating from single clones were stably cultured out of the urine sediment of KTX recipients with suspected BKPyVAN. The cell morphology, polyploidy, virus replication and protein production were investigated by confocal microscopy using both a monoclonal (mAb 4942) and a polyclonal rabbit anti-VP1-specific antibody (RantiVP1 Ab). Immunoblotting was performed to investigate changes in the VP1 protein. Both antibodies visualized VP1 and the mAb 4942 recognized VP1 in cytoplasmic vesicles exhibiting idiomorphic sizes when released from the cells. In contrast, the polyclonal antibody detected VP1 within the nucleus and in cytoplasm in colocalization with the endoplasmic reticulum marker CNX. At the nuclear rim, VP1 was recognized by both antibodies. Immunoblotting revealed two smaller versions of VP1 in urinary decoy cell extracts, potentially from different translation start sites as evaluated by in silico analysis. Oxford Nanopore sequencing showed integration of BKPyV DNA in chromosomes 3, 4 and 7 in one of the five tested primary cell lines which produced high viral copies throughout four passages before transcending into senescence. The different staining with two VP1-specific antibodies emphasizes the modification of VP1 during the process of virus maturation and cellular exit. The integration of BKPyV into the human genome leads to high virus production; however, this alone does not transform the cell line into a permanently cycling and indefinitely replicating one.

Aim: To evaluate the fracture resistance and failure mode of endodontically treated maxillary 1stpremolar restored with two restorative techniques (direct inlay composite & ceramic inlay). Methods: Forty extracted maxillary 1st premolars for orthodontic reasons were divided into 4 groups. GROUP 1 (Positive control) GROUP 2 (Negative control) GROUP 3 (Composite Resin Direct Inlay) It contains premolars including standard mesio – occluso – distal (MOD) cavity preparation after doing the endodontic treatment. The MOD cavities were filled with Composite Resin by direct technique fabricated from 3M Filtek Z350 XT Universal Restorative Syringe, shade A3 and using 3M ESPE Single bond universal according to manufactures instructions, and GROUP 4 (Ceramic Inlay) It contains premolars including standard mesio – occluso – distal (MOD) cavity preparation after doing endodontic treatment. construction of Ceramic Inlay by indirect technique from IPS e.max® CAD HT, shade A3 according to manufactures instructions. and cemented by DENTSPLY Calibra Universal Dual Cure Auto Mix Syringe Refill. Results: Showed that the premolars in Group 1 (positive control-intact premolars) exhibited fracture resistance results 870.9 ± 230.13 which were much greater than those in the remaining groups, whereas those in Group 2 (negative control-unfilled MOD cavity) exhibited the worst results 254.02 ± 80.45 (p < 0.05). The fracture resistance of premolars in Group 3 (composite resin) 593.8 ± 130.25, and the Group 4 (ceramic inlay) 658.17 ± 140.89 was statistically equivalent p > 0.05 (non-significant), while in comparing Group 3&4 with unrestored teeth (Group 2) they are showing significantly higher fracture resistance results than group 2 (p < 0.05). The failure mode results showed that Group 1 (intact teeth) showed the greatest restorable fractures. In Group 2 (negative control-unrestored MOD cavity), there had been no statistically significant variation based on fracture modes. Group 4 (ceramic inlay) showed a significant degree of irreparable fracture (p < 0.05). Conclusion: The resting sound occlusal tooth structure increases the fracture resistance of teeth that have undergone endodontic treatment, and the fracture mode is repairable. The occlusal final restoration raises the fracture resistance of root canal treated teeth, but the stiff one produces unfavourable failure mode.