The increase in multidrug resistant bacteria has sparked an interest in the development of novel antibiotics. Antimicrobial peptides (AMPs) that operate by crossing the cell membrane may also have the potential to deliver drugs to intracellular targets. Buforin 2 (BF2) is an AMP that shares sequence identity with a fragment of histone subunit H2A and whose bactericidal mechanism depends on membrane translocation and DNA binding. Previously, novel histone-derived antimicrobial peptides (HDAPs) were designed based on properties of BF2, and DesHDAP1 and DesHDAP3 showed significant antibacterial activity. In this study, their DNA binding, permeabilization, and translocation abilities were assessed independently and compared to antibacterial activity to determine whether they share a mechanism with BF2. To investigate the importance of proline in determining the peptides' mechanisms of action, proline to alanine mutants of the novel peptides were generated. DesHDAP1, which shows significant similarities to BF2 in terms of secondary structure, translocates effectively across lipid vesicle and bacterial membranes, while the DesHDAP1 proline mutant shows reduced translocation abilities and antimicrobial potency. In contrast, both DesHDAP3 and its proline mutant translocate poorly, though the DesHDAP3 proline mutant is more potent. Our findings suggest that a proline hinge can promote membrane translocation in some peptides, but that the extent of its effect on permeabilization depends on the peptide's amphipathic properties. Our results also highlight the different antimicrobial mechanisms exhibited by histone-derived peptides and suggest that histones may serve as a source of novel AMPs with varied properties.

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Background Infections are common in advanced dementia. Little is known about healthcare proxy involvement in decision making regarding infections. Design Prospective cohort study Setting/Participants 362 nursing home (NH) residents with advanced dementia and their proxies in 35 Boston-area facilities. Measurements Charts were abstracted monthly (up to 12 months) for documentation of suspected infections and provider/proxy discussions for each episode. Proxies were interviewed within 8 weeks of the infection to determine their awareness and decision-making involvement. Factors associated with proxy awareness and discussion documentation were identified. Results There were 496 suspected infections; proxies were reached for interview for 395 (80%). Proxy/provider discussions were documented for 207/395 episodes (52%), yet proxies were aware of only 156/395 (39%). Proxies participated in decision-making for 89/156 (57%) episodes of which they were aware. Proxy awareness was associated with antimicrobial use (adjusted odds ratio (AOR) 3.43, 95% confidence interval (CI) 1.94–6.05), hospital transfer (AOR 3.00, 95% CI 1.19–7.53), infection occurrence within 30 days of death (AOR 3.32, 95% CI 1.54–7.18) and fewer days between infection and study interview (AOR 2.71, 95% CI 1.63–4.51). Discussion documentation was associated with the resident residing in a dementia special care unit (AOR 1.71, 95% CI 1.04–2.80), not on hospice (AOR 3.25, 95% CI 1.31–8.02), more provider visits (AOR 1.71, 95% CI 1.07–2.75), proxy visits > 7 hours/week (AOR 1.93, 95% CI 1.02–3.67), and episode within 30 days of death (AOR 3.99, 95% CI 1.98–8.02). Conclusion Proxies are unaware of and do not participate in decision-making for most suspected infections experienced by NH residents with advanced dementia. Proxy awareness of episodes and documentation of provider/proxy discussions are not congruent.

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs).9-13 LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

Introduction Alcohol use disorder (AUD) is commonly undertreated. Physicians cite discomfort with AUD medication as a barrier to treatment. While several curricula teach and assess screening and brief interventions, few teach and assess learner knowledge of treatment options. Methods We created a video- and case-based curriculum for internal medicine residents delivered by 16 internal medicine faculty in three 30-minute sessions at four clinic sites. Learner knowledge, attitudes, and confidence were assessed before and after the curriculum. We used qualitative methods to evaluate learner reflections. We also assessed faculty satisfaction with the curriculum. Results Of 153 residents receiving the curriculum, 35 (23%) completed both pre- and postsurveys. Median percent correct on knowledge questions improved from 67% pre- to 80% postcurriculum ( p < .001). Confidence increased for all three items assessing it, with a notable increase in confidence with pharmacotherapy (2.9 pre- vs. 4.5 postcurriculum on a 7-point Likert scale with high scores indicating greater confidence, p < .001). Positive attitudes toward people with AUD increased from 3.4 pre- to 3.9 postcurriculum ( p < .001) on a 7-point Likert scale. Learners continued to express concerns about prescribing logistics, the role of primary care, and management of ongoing use. Thirteen of 16 faculty (83%) completed the postcurricular survey; all said they would be happy to facilitate again. Discussion Implementation of this curriculum for the management of AUD improved resident knowledge, attitudes, and confidence in AUD treatment. The curriculum was acceptable to faculty and is ideal for programs looking to expand teaching about AUD.