Cationic Peptide Exposure Enhances Pulsed-Electric-Field-Mediated Membrane Disruption

Background: The use of pulsed electric fields (PEFs) to irreversibly electroporate cells is a promising approach for destroying undesirable cells. This approach may gain enhanced applicability if the intensity of the PEF required to electrically disrupt cell membranes can be reduced via exposure to a molecular deliverable. This will be particularly impactful if that reduced PEF minimally influences cells that are not exposed to the deliverable. We hypothesized that the introduction of charged molecules to the cell surfaces would create regions of enhanced transmembrane electric potential in the vicinity of each charged molecule, thereby lowering the PEF intensity required to disrupt the plasma membranes. This study will therefore examine if exposure to cationic peptides can enhance a PEF’s ability to disrupt plasma membranes. Methodology/Principal Findings We exposed leukemia cells to 40 μs PEFs in media containing varying concentrations of a cationic peptide, polyarginine. We observed the internalization of a membrane integrity indicator, propidium iodide (PI), in real time. Based on an individual cell’s PI fluorescence versus time signature, we were able to determine the relative degree of membrane disruption. When using 1–2 kV/cm, exposure to >50 μg/ml of polyarginine resulted in immediate and high levels of PI uptake, indicating severe membrane disruption, whereas in the absence of peptide, cells predominantly exhibited signatures indicative of no membrane disruption. Additionally, PI entered cells through the anode-facing membrane when exposed to cationic peptide, which was theoretically expected. Conclusions/Significance: Exposure to cationic peptides reduced the PEF intensity required to induce rapid and irreversible membrane disruption. Critically, peptide exposure reduced the PEF intensities required to elicit irreversible membrane disruption at normally sub-electroporation intensities. We believe that these cationic peptides, when coupled with current advancements in cell targeting techniques will be useful tools in applications where targeted destruction of unwanted cell populations is desired

The neighbourhood environment and use of neighbourhood resources in older adults with and without lower limb osteoarthritis:results from the Hertfordshire Cohort Study

This study aimed to examine the associations of perceptions of neighbourhood cohesion and neighbourhood problems and objectively measured neighbourhood deprivation with the use of neighbourhood resources by older adults with and without lower limb osteoarthritis (LLOA), and to assess whether these relationships are stronger in older persons with LLOA than in those without the condition. Data from the Hertfordshire Cohort Study were used. American College of Rheumatology classification criteria were used to diagnose clinical LLOA (knee and/or hip osteoarthritis). Use of neighbourhood resources was assessed using the Home and Community Environment instrument. Participants were asked about their perceptions of neighbourhood cohesion and neighbourhood problems. Objective neighbourhood deprivation was assessed using the Index of Multiple Deprivation score based on 2010 census data. Of the 401 participants (71–80 years), 74 (18.5 %) had LLOA. The neighbourhood measures were not significantly associated with use of resources in the full sample. A trend for a negative association between use of public transport and perceived neighbourhood problems was observed in participants with LLOA (OR = 0.77, 99 % CI = 0.53–1.12), whereas a trend for a positive association between perceived neighbourhood problems and use of public transport was found in participants without LLOA (OR = 1.18, 99 % CI = 1.00–1.39). The perception of more neighbourhood problems seems only to hinder older adults with LLOA to make use of public transport. Older adults with LLOA may be less able to deal with neighbourhood problems and more challenging environments than those without the condition

Predictors of burnout, work engagement and nurse reported job outcomes and quality of care: a mixed method study

BACKGROUND: High levels of work-related stress, burnout, job dissatisfaction, and poor health are common within the nursing profession. A comprehensive understanding of nurses’ psychosocial work environment is necessary to respond to complex patients’ needs. The aims of this study were threefold: (1) To retest and confirm two structural equation models exploring associations between practice environment and work characteristics as predictors of burnout (model 1) and engagement (model 2) as well as nurse-reported job outcome and quality of care; (2) To study staff nurses’ and nurse managers’ perceptions and experiences of staff nurses’ workload; (3) To explain and interpret the two models by using the qualitative study findings. METHOD: This mixed method study is based on an explanatory sequential study design. We first performed a cross-sectional survey design in two large acute care university hospitals. Secondly, we conducted individual semi-structured interviews with staff nurses and nurse managers assigned to medical or surgical units in one of the study hospitals. Study data was collected between September 2014 and June 2015. Finally, qualitative study results assisted in explaining and interpreting the findings of the two models. RESULTS: The two models with burnout and engagement as mediating outcome variables fitted sufficiently to the data. Nurse-reported job outcomes and quality of care explained variances between 52 and 62%. Nurse management at the unit level and workload had a direct impact on outcome variables with explained variances between 23 and 36% and between 12 and 17%, respectively. Personal accomplishment and depersonalization had an explained variance on job outcomes of 23% and vigor of 20%. Burnout and engagement had a less relevant direct impact on quality of care (≤5%). The qualitative study revealed various themes such as organisation of daily practice and work conditions; interdisciplinary collaboration, communication and teamwork; staff nurse personal characteristics and competencies; patient centeredness, quality and patient safety. Respondents’ statements corresponded closely to the models’ associations. CONCLUSION: A deep understanding of various associations and impacts on studied outcome variables such as risk factors and protective factors was gained through the retested models and the interviews with the study participants. Besides the softer work characteristics — such as decision latitude, social capital and team cohesion — more insight and knowledge of the hard work characteristic workload is essential

Associations between perceived neighbourhood problems and quality of life in older adults with and without osteoarthritis:Results from the Hertfordshire cohort study

This study examined whether the association of quality of life (QoL) with perceived neighbourhood problems is stronger in older adults with osteoarthritis (OA) than in those without OA. Of all 294 participants, 23.8% had OA. More perceived neighbourhood problems were associated with a stronger decrease in QoL over time in participants with OA (B=-0.018; p=0.02) than in those without OA (B=-0.004; p=0.39). Physical activity did not mediate this relationship. Older adults with OA may be less able to deal with more challenging environments

A timeline summarizing the experimental protocol.

<p>The microcuvette was treated with an adhesion peptide so that cells would settle into the microchannel and remain immobilized during experimentation. Cells and PI were added to the microcuvette 300 seconds prior to PEF exposure and allowed to settle into the microchannel. If peptides were used, at 60 seconds before PEF exposure, they were added at twice their desired concentration so that, when combined with the cell/PI mixture in a 1∶1 ratio, they would dilute into the desired experimental concentration. Also at 60 seconds prior to PEF exposure, fluorescence imaging of the cells began (1 image every 10 seconds). At time 0, a PEF was administered and cells were imaged for 1400 seconds.</p

When used in concert with an externally applied PEF, cationic peptides can be used to enhance membrane disruption at lower PEF intensities at the anode-facing cell membrane.

<p>When a cell rests in an ionic medium containing membrane impermeable entities (<b><i>A</i></b>) and cationic peptides are added to that medium (<b><i>B</i></b>), a PEF can be used to accumulate those cationic peptides about the anode-facing membrane (<b><i>C</i></b>) where the anode-facing membrane can experience a local increases in electrostatic potential near individual peptides (<i>V</i>_p) plus enhanced electrostatic potential due to the ion relocation due to the applied field (<i>V</i>_e). These local enhancements in transmembrane electrostatic potentials can result in anodally preferenced electroporation at lower PEF intensities (<b><i>D</i></b>) and subsequent internalization of normally membrane-impermeable entities through the anode-facing membrane (<b><i>E</i></b>).</p

Cationic peptide exposure alone influenced membrane permeability to PI.

<p><b><i>A</i></b>: Cells were exposed to a peptides composed of neutral residues at neutral pH (polyasparagine, at left) and peptides with cationic residues at neutral pH (polyarginine, at right). These two peptides were of the same molecular weight. When 250 μg/ml of neutral polyasparagine or cationic polyarginine was added to cell media at −60 seconds, cells either fluoresced at baseline levels (<b><i>B</i></b>) or internalized PI in delayed, accelerating, and high levels (<b><i>C</i></b>). <b><i>D</i></b>–<b><i>G</i></b>: Reducing the cationic peptide concentration reduced the number of cells that internalized PI during the course of the experiment, and increased the delay time observed for abrupt, accelerated, high-level PI uptake. In <b><i>B</i></b>–<b><i>G</i></b>, the number of cells examined was 15, 18, 16, 14, 11, and 22, respectively.</p

Cationic peptide exposure can eventually lead to prolonged membrane disruption through electrostatic peptide-membrane interaction.

<p>A cell rests in an ionic media with membrane-impermeable entities (<b><i>A</i></b>) and cationic peptide is introduced into that media (<b><i>B</i></b>). Cationic peptides will electrostatically collect about the negatively charged plasma membrane (<b><i>C</i></b>) and increase the electrostatic potential across the membrane in the vicinity of the peptide (<i>V</i>_p). <b><i>D</i></b>–<b><i>E</i></b>: These transmembrane voltages may be sufficient for electroporation and disrupt the membrane in a manner leading to internalization of normally membrane-impermeable entities. <b><i>E</i></b>–<b><i>F</i></b>: Particularly at higher cationic peptide concentrations, the media serves as a reservoir for additional peptides to co-localize with the plasma membrane, causing prolonged membrane permeability.</p