Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase-transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals. Copyright (C) 2004 John Wiley Sons, Ltd.

Gene transfer into genomes of human cells by the sleeping beauty transposon system

The Sleeping Beauty (SB) transposon system, derived from teleost fish sequences, is extremely effective at delivering DNA to vertebrate genomes, including those of humans. We have examined several parameters of the SB system to improve it as a potential, nonviral vector for gene therapy. Our investigation centered on three features: the carrying capacity of the transposon for efficient integration into chromosomes of HeLa cells, the effects of overexpression of the SB transposase gene on transposition rates, and improvements in the activity of SB transposase to increase insertion rates of transgenes into cellular chromosomes. We found that SB transposons of about 6 kb retained 50% of the maximal efficiency of transposition, which is sufficient to deliver 70-80% of identified human cDNAs with appropriate transcriptional regulatory sequences. Overexpression inhibition studies revealed that there are optimal ratios of SB transposase to transposon for maximal rates of transposition, suggesting that conditions of delivery of the two-part transposon system are important for the best gene-transfer efficiencies. We further refined the SB transposase to incorporate several amino acid substitutions, the result of which led to an improved transposase called SB11. With SB11 we are able to achieve transposition rates that are about 100-fold above those achieved with plasmids that insert into chromosomes by random recombination. With the recently described improvements to the transposon itself, the SB system appears to be a potential gene-transfer tool for human gene therapy.

Structure-function analysis of the inverted terminal repeats of the Sleeping Beauty transposon

Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates. (C) 2002 Elsevier Science Ltd. All rights reserved.

Sleeping site selection by the golden-handed tamarin Saguinus midas midas: The role of predation risk, proximity to feeding sites, and territorial defence

Tamarin monkeys, of the genus Saguinus, spend over half their lives at arboreal sleeping sites. The decision as to which site to use is likely to have considerable fitness consequences. These decisions about sleeping sites by three troops of golden-handed tamarin Saguinus midas midas were examined over a 9-mo period at a rainforest site in French Guiana. Data are presented on the physical nature of sleeping sites, their number, position within home ranges, and pattern of use and reuse, aspects of behaviour at retirement and egress, and predation attempts on the study troops. Cumulative plot analysis indicated that a tamarin troop used 30-40 sleeping sites in a 100-day period, approximately half of which were used very infrequently, so that consecutive reuse was never greater than three nights. Sleeping trees were superior in architectural parameters and liana weight to non-sleeping trees. There were no more sleeping sites than expected within the home range boundary region of the tamarins or in areas of overlap with the home ranges of neighbouring troops. Tamarins selected sleeping sites nearest to the last feeding site of the day on 25% of occasions. The study troops engaged in a number of activities that may reduce predation risk; raptor attacks on the study troops over 9 mo were frequent but unsuccessful. Tamarins often visited a sleeping site several hours before arrival, and were more likely to visit a site before use if they had not used it recently. The decision to select a sleeping site therefore involved knowledge of the previous frequency of use of that site.

Fairground Music; Augustine Sleeping Before He Can Talk; Telegraph; Cathedral

Four poems.

Music for Sleeping & Waking Minds

Music for Sleeping & Waking Minds is a 8-hour composition intended for overnight listening. It features 4 performers who wear custom-designed EEG sensors. The performers rest and fall asleep as they naturally would. Over the course of one night, their brainwave activity generates a spatial audio environment. Audiences are invited to sleep or listen as they wish. Composition & concept by Gascia Ouzounian. Physiological interface and interaction design by R. Benjamin Knapp. Audio interface and interaction design by Eric Lyon.

Do psychosocial factors moderate the association of fixed-term employment with work interference with family and sleeping problems in registered nurses: A cross-sectional questionnaire survey

Background: The use affixed-term employment has increased lately, particularly in Europe and in the health care sector. Previous studies have shown that especially among the health care sector employee's organizational justice perceptions and job control are important factors that are directly related to the welfare and attitudes of employees and may also help to buffer the negative impacts of many detrimental factors.

The Association of Distress and Sleeping Problems With Physicians' Intentions To Change Profession: The Moderating Effect of Job Control

The present study examined whether job control moderated the association between stress indicators (distress and sleeping problems) and intentions to change profession among 2,650 Finnish physicians. Ordinal logistic regression analysis was applied. The authors found that high levels of distress and sleeping problems were associated with higher levels of intentions to change profession, whereas high job control was associated with lower levels of intentions to change profession even after adjusting for the effects of gender, age, and employment sector. In addition, high job control was able to mitigate the positive association that distress and sleeping problems had with intentions to change profession. Our findings highlight the importance of offering more job control to physicians to prevent unnecessary physician turnover.

The molecular dynamics of Trypanosoma brucei UDP-galactose 4'-epimerase:a drug target for African sleeping sickness

During the past century, several epidemics of human African trypanosomiasis, a deadly disease caused by the protist Trypanosoma brucei, have afflicted sub-Saharan Africa. Over 10 000 new victims are reported each year, with hundreds of thousands more at risk. As current drug treatments are either highly toxic or ineffective, novel trypanocides are urgently needed. The T. brucei galactose synthesis pathway is one potential therapeutic target. Although galactose is essential for T. brucei survival, the parasite lacks the transporters required to intake galactose from the environment. UDP-galactose 4'-epimerase (TbGalE) is responsible for the epimerization of UDP-glucose to UDP-galactose and is therefore of great interest to medicinal chemists. Using molecular dynamics simulations, we investigate the atomistic motions of TbGalE in both the apo and holo states. The sampled conformations and protein dynamics depend not only on the presence of a UDP-sugar ligand, but also on the chirality of the UDP-sugar C4 atom. This dependence provides important insights into TbGalE function and may help guide future computer-aided drug discovery efforts targeting this protein.

To be inside someone else's dream: On Music for Sleeping & Waking Minds

Music for Sleeping & Waking Minds (2011-2012) is a new,overnight work in which four performers fall asleep while wearing custom designed EEG sensors which monitor their brainwave activity. The data gathered from the EEG sensors is applied in real time to different audio and image signal processing functions, resulting in continuously evolving multichannel sound environment and visual projection. This material serves as an audiovisual description of the individual and collective neuro physiological state of the ensemble. Audiences are invited to experience the work in different states of attention: while alert and asleep, resting and awakening.

Sleeping beauty.

La princesa Aurora se pincha en un dedo con una rueca y cae en un profundo sueño. El príncipe Philip acude enseguida a su rescate, podrá un beso a la bella princesa despertarla, ó dormirá para siempre.

Who's been sleeping in my porridge? : a book of wacky poems and pictures.

Colección de poemas absurdos sobre criaturas de todas formas y tamaños:extraterrestres, gigantes, dinosaurios, monstruos, bebés, y por supuesto, un montón de niños.

Sleeping with half open eyes: dreams and realities in The Cry of the Owl

This article is a close analysis of The Cry of the Owl (Thraves, 2009). It is also part of larger project to bring together traditions of detailed criticism with those of production history, which culminates in second article on the film due to be published in 2011. The detail of the argument concerns analysing a range of the film’s key signifying systems, with a particular interest in the way the film explores the gap between images / impressions and characters’ realities; engages in a complex way with generic traditions and modes of address; establishes complex patterns of connection and contrast through blocking, camera strategies and narrative structure.