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Expanding the Versatility of Phage Display I: Efficient Display of Peptide-Tags on Protein VII of the Filamentous Phage

Background: Phage display is a platform for selection of specific binding molecules and this is a clear-cut motivation for increasing its performance. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII), or the minor coat protein III (pIII). Display on other coat proteins such as pVII allows for display of heterologous peptide sequences on the virions in addition to those displayed on pIII and pVIII. In addition, pVII display is an alternative to pIII or pVIII display. Methodology/Principal Findings: Here we demonstrate how standard pIII or pVIII display phagemids are complemented with a helper phage which supports production of virions that are tagged with octa FLAG, HIS6 or AviTag on pVII. The periplasmic signal sequence required for pIII and pVIII display, and which has been added to pVII in earlier studies, is omitted altogether. Conclusions/Significance: Tagging on pVII is an important and very useful add-on feature to standard pIII and pVII display. Any phagemid bearing a protein of interest on either pIII or pVIII can be tagged with any of the tags depending simply on choice of helper phage. We show in this paper how such tags may be utilized for immobilization and separation as well as purification and detection of monoclonal and polyclonal phage populations. © 2011 Wälchli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

FIP1/RCP Binding to Golgin-97 Regulates Retrograde Transport from Recycling Endosomes to the trans-Golgi Network

This study shows that Rab11 and its binding protein FIP1 are required for retrograde delivery of TGN38 and Shiga toxin from early/recycling endosomes to the TGN. We also demonstrate that Golgin-97 as a FIP1-binding protein and that this binding regulates the targeting of retrograde transport vesicles to the TGN

Retrograde traffic in the biosynthetic-secretory route

In the biosynthetic-secretory route from the rough endoplasmic reticulum, across the pre-Golgi intermediate compartments, the Golgi apparatus stacks, trans Golgi network, and post-Golgi organelles, anterograde transport is accompanied and counterbalanced by retrograde traffic of both membranes and contents. In the physiologic dynamics of cells, retrograde flow is necessary for retrieval of molecules that escaped from their compartments of function, for keeping the compartments’ balances, and maintenance of the functional integrities of organelles and compartments along the secretory route, for repeated use of molecules, and molecule repair. Internalized molecules may be transported in retrograde direction along certain sections of the secretory route, and compartments and machineries of the secretory pathway may be misused by toxins. An important example is the toxin of Shigella dysenteriae, which has been shown to travel from the cell surface across endosomes, and the Golgi apparatus en route to the endoplasmic reticulum, and the cytosol, where it exerts its deleterious effects. Most importantly in medical research, knowledge about the retrograde cellular pathways is increasingly being utilized for the development of strategies for targeted delivery of drugs to the interior of cells. Multiple details about the molecular transport machineries involved in retrograde traffic are known; a high number of the molecular constituents have been characterized, and the complicated fine structural architectures of the compartments involved become more and more visible. However, multiple contradictions exist, and already established traffic models again are in question by contradictory results obtained with diverse cell systems, and/or different techniques. Additional problems arise by the fact that the conditions used in the experimental protocols frequently do not reflect the physiologic situations of the cells. Regular and pathologic situations often are intermingled, and experimental treatments by themselves change cell organizations. This review addresses physiologic and pathologic situations, tries to correlate results obtained by different cell biologic techniques, and asks questions, which may be the basis and starting point for further investigations

HDAC2 depletion promotes osteosarcoma's stemness both in vitro and in vivo: a study on a putative new target for CSCs directed therapy

Background: Cancer stem cells (CSCs) play a key role in cancer initiation, progression and chemoresistance. Epigenetic alterations have been identified as prominent factors that contribute to the CSCs phenotype. Here, we investigated the effects of the HDAC inhibitor valproic acid (VPA) and the demethylating agent, 5’azacytidine (DAC) on the stem phenotype of MG63 and Saos2 osteosarcoma cell lines. Methods: Saos2 and MG63 cells were treated with DAC and VPA, alone and in combination. Untreated and treated cells were examined for stemness phenotype by cytometry and real-time PCR. Sarcospheres and colonies formation were also evaluated. Moreover, histone modification and methylation were tested by flow cytomery and western blotting. HDAC2 depleted cells were examined for stemness phenotype and their ability to generate tumors in NOD/SCID IL2R-gamma-0 (NSG) mice. HDAC2 expression on human osteosarcoma tissues was evaluated. Results: We found that DAC and VPA induce an increased expression of stem markers including CD133, OCT4, SOX2 and NANOG, and an increased ability in sarcospheres and colonies formation efficiency. Interestingly, we showed that DAC and VPA treatment decreased repressive histone markers, while increased the active ones. These histone modifications were also associated with an increase of acetylation of histones H3, a decrease of DNA global methylation, HDAC2 and DNMT3a. Furthermore, HDAC2 silenced-MG63 and Saos2 cells acquired a stem phenotype, and promoted in vivo tumorigenesis. In human osteosarcoma tissues, HDAC2 was strongly expressed in nucleus. Conclusions: Collectively, our results suggest that VPA and DAC induce an expansion of osteosarcoma CSCs, and we report for the first time that HDAC2 is a key factor regulating both CSCs phenotype and in vivo cancer growth. In conclusion, we have identified HDAC2 as a potential therapeutic target in human osteosarcoma treatment

Forebygging av trykksår

Oppdraget ble gitt av Nasjonalt kunnskapssenter for helsetjenesten, sekretariatet for pasientsikkerhetskampanjen 2011. Hovedkonklusjoner (gradert vitenskapelig kunnskapsgrunnlag) Forekomsten av trykksår kan reduseres ved bruk av trykkavlastende madrasser (høy kvalitet), puter eller saueskinnspels (moderat kvalitet). Det er utilstrekkelig kunnskapsgrunnlag til å vurdere effekt av å bruke risikoscoringsverktøy for utvikling av trykksår. Risikovurdering for utvikling av trykksår er anbefalt i nasjonale og internasjonale veiledere og retningslinjer. Der er utilstrekkelig kunnskapsgrunnlag til å bedømme effekt av å observere hud for å forebygge trykksår. Bruk av pH balansert såpe kan redusere forekomsten av trykksår (lav kvalitet). Der er usikkert om tilleggsernæring kan forebygge trykksår (svært lav kvalitet)

Clinical studies of solvent exposed workers. A systematic search for literature

I Norge er det enighet om de overordnede kriteriene for å gi diagnosen løsemiddelskade. Likevel er det ikke utviklet en enhetlig og detaljert modell for utredning av slik skade. Nasjonalt kunnskapssenter for helsetjenesten har i samarbeid med de norske utredningsmiljøene for løsemiddelskade gjennomført et litteratursøk etter kliniske studier av personer med langvarig yrkesbetinget løsemiddeleksponering. Vi har sortert publikasjonene etter studiedesign, populasjon, type undersøkelse og studiespørsmål. Vi har ikke rapportert utfall eller vurdert kvaliteten av studiene.Clinical studies of solvent exposed workers. A systematic search for literatur