Comparison of lentiviral vector titration methods

BACKGROUND: Lentiviral vectors are efficient vehicles for stable gene transfer in dividing and non-dividing cells. Several improvements in vector design to increase biosafety and transgene expression, have led to the approval of these vectors for use in clinical studies. Methods are required to analyze the quality of lentiviral vector production, the efficiency of gene transfer and the extent of therapeutic gene expression. RESULTS: We compared lentiviral vector titration methods that measure pg p24/ml, RNA equivalents/ml, transducing units (TU/ml) or mRNA equivalents. The amount of genomic RNA in vector particles proves to be reliable to assess the production quality of vectors encoding non-fluorescent proteins. However, the RNA and p24 titers of concentrated vectors are rather poor in predicting transduction efficiency, due to the high variability of vector production based on transient transfection. Moreover, we demonstrate that transgenic mRNA levels correlate well with TU and can be used for functional titration of non-fluorescent transgenes. CONCLUSION: The different titration methods have specific advantages and disadvantages. Depending on the experimental set-up one titration method should be preferred over the others

Structure, mechanics, and binding mode heterogeneity of LEDGF/p75-DNA nucleoprotein complexes revealed by scanning force microscopy

LEDGF/p75 is a transcriptional coactivator implicated in the pathogenesis of AIDS and leukemia. In these contexts, LEDGF/p75 acts as a cofactor by tethering protein cargo to transcriptionally active regions in the human genome. Our study - based on scanning force microscopy (SFM) imaging - is the first to provide structural information on the interaction of LEDGF/p75 with DNA. Two novel approaches that allow obtaining insights into the DNA conformation inside nucleoprotein complexes revealed (1) that LEDGF/p75 can bind at least in three different binding modes, (2) how DNA topology and protein dimerization affect these binding modes, and (3) geometrical and mechanical aspects of the nucleoprotein complexes. These structural and mechanical details will help us to better understand the cellular mechanisms of LEDGF/p75 as a transcriptional coactivator and as a cofactor in disease

‘Promoting and preserving safety and a life‐oriented perspective’ : a qualitative study of nurses' interactions with patients experiencing suicidal ideation

Suicide prevention is an important imperative in psychiatric hospitals, where nurses have a crucial role in and make essential contributions to suicide prevention and promoting the recovery of patients experiencing suicidal ideation. The present qualitative grounded theory study aimed to uncover and understand the actions and aims of nurses in psychiatric hospitals during their interactions with patients experiencing suicidal ideation. Interviews were conducted with 26 nurses employed on 12 wards in four psychiatric hospitals. The data analysis was inspired by the Qualitative Analysis Guide of Leuven. The findings show that nurses' actions and aims in their interactions with patients experiencing suicidal ideation are captured in the core element 'promoting and preserving safety and a life-oriented perspective'. This core element represents the three interconnected elements 'managing the risk of suicide', 'guiding patients away from suicidal ideation', and 'searching for balance in the minefield'. The enhanced understanding of nurses' actions and aims can inform concrete strategies for nursing practice and education. These strategies should aim to challenge overly controlling and directing nursing approaches and support nurses' capacity and ability to connect and collaborate with patients experiencing suicidal ideation