Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background: Tight junctions are an intercellular adhesion complex of epithelial and endothelial cells, and form a paracellular barrier that restricts the diffusion of solutes on the basis of size and charge. Tight junctions are formed by multiprotein complexes containing cytosolic and transmembrane proteins. How these components work together to form functional tight junctions is still not well understood and will require a complete understanding of the molecular composition of the junction. Results: Here we identify a new transmembrane component of tight junctions: MarvelD3, a four-span transmembrane protein. Its predicted transmembrane helices form a Marvel (MAL and related proteins for vesicle traffic and membrane link) domain, a structural motif originally discovered in proteins involved in membrane apposition and fusion events, such as the tight junction proteins occludin and tricellulin. In mammals, MarvelD3 is expressed as two alternatively spliced isoforms. Both isoforms exhibit a broad tissue distribution and are expressed by different types of epithelial as well as endothelial cells. MarvelD3 co-localises with occludin at tight junctions in intestinal and corneal epithelial cells. RNA interference experiments in Caco-2 cells indicate that normal MarvelD3 expression is not required for the formation of functional tight junctions but depletion results in monolayers with increased transepithelial electrical resistance. Conclusions: Our data indicate that MarvelD3 is a third member of the tight junction-associated occludin family of transmembrane proteins. Similar to occludin, normal expression of MarvelD3 is not essential for the formation of functional tight junctions. However, MarvelD3 functions as a determinant of epithelial paracellular permeability properties

Postal card from Charles E. Steed

Postal card concerning winter courses at Utah Agricultural College

A Phenomenological Study of Graduated Nursing Student Athletes\u27 Experiences Balancing Academics and Athletics

The purpose of this transcendental phenomenological study is to describe the lived experiences of eleven graduated nursing student athletes who completed traditional, four-year nursing programs while concurrently finishing four years of athletic eligibility in their respective sport at three private, Christian, Midwest universities and across three different competitive collegiate athletic divisions. The theories guiding this study are Tinto’s Theory of Individual Departure from Institutions of Higher Learning, Astin’s Theory of Student Involvement, and Lazarus and Folkman’s Transactional Model of Stress Response, as they relate to student athletes’ persistence to graduation and to nursing students who reportedly experience higher levels of stress than other college students. Participants were purposefully selected to answer the following: How do graduated nursing student athletes describe their experiences in balancing sports and academics while completing a traditional, four-year nursing program and participating in intercollegiate sports? Data collection was conducted using journaling, semi-structured individual interviews, and focus groups. Confidentiality was maintained by using pseudonyms for all colleges and participants. Data analysis was conducted via pattern, theme, and content analysis. Validity and trustworthiness were established via expert and member reviews, as well as triangulation of participant groups, data sources, audit trails, enumeration tables, and inclusion of participant quotes

Functional analysis of MarvelD3, a novel transmembrane protein of the tight junction

Tight junctions are an intercellular adhesion complex of epithelial and endothelial cells. They form a paracellular diffusion barrier and interact with a network of intracellular signalling mechanisms that control junction function, gene expression and cell behaviour. Tight junctions are formed by multiprotein complexes containing cytosolic and transmembrane proteins. In this thesis I have identified a novel fourpass transmembrane protein of the tight junction called MarvelD3 and begun to analyse its function in the regulation of intracellular signalling pathways from the junction. There are two isoforms of MarvelD3, both of which show a broad tissue distribution and are expressed in different types of epithelial and endothelial cells. MarvelD3 co-localises with occludin at the tight junction in epithelial cells. I have found that MarvelD3 is not necessary for junction formation, but may have a role in the regulation of ion conductance properties of the tight junction. Functional analyses combining loss- and gain-of-function approaches in epithelial cell lines have further identified a role for MarvelD3 in the regulation of cell proliferation, migration and the cellular response to hyperosmotic shock. MarvelD3 expression regulates levels of active c-Jun N-terminal kinase (JNK) and AP1 signalling, possibly via an interaction between its N-terminus and the MAP kinase kinase kinase MEKK1. I have also shown MarvelD3 to be implicated in regulation of the actin cytoskeleton, affecting leading edge formation in migrating cells and cytoskeletal rearrangements in response to hyperosmotic shock. I will also describe some initial studies conducted in Xenopus laevis embryos in which depletion of Xenopus MarvelD3 by morpholino injection results in curvature of the anterioposterior axis and reduced pigmentation, possibly resulting from a defect in neural crest cell migration

Impact of Dose De-Escalation and Escalation on Daptomycin’s Pharmacodynamics against Clinical Methicillin-Resistant Staphylococcus aureus Isolates in an In Vitro Model

De-escalation and escalation therapeutic strategies are commonly employed by clinicians on the basis of susceptibility results and patient response. Since no in vitro or in vivo data are currently available to support one strategy over the other for daptomycin, we attempted to evaluate the effects of dose escalation and de-escalation on daptomycin activity against methicillin-resistant Staphylococcus aureus (MRSA) isolates using an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model with simulated endocardial vegetations. Three clinical MRSA isolates, including one heterogeneous vancomycin-intermediate S. aureus (hVISA) isolate and one vancomycin-intermediate S. aureus (VISA) isolate, were exposed to daptomycin at 10 or 6 mg/kg of body weight/day for 8 days using a starting inoculum of 109 CFU/g of vegetations, with dose escalation and de-escalation initiated on the fourth day. Daptomycin MIC values ranged from 0.5 to 1 g/ml. In the PK/PD model, high-dose daptomycin (10 mg/kg/day) and de-escalation simulation (10 to 6 mg/kg/day) appeared to be the most efficient regimens against the three tested isolates, exhibiting the fastest bactericidal activity (4 to 8 h) compared to that of the standard regimen of 6 mg/kg/day and the escalation therapy of 6 to 10 mg/kg/day. The differences in the numbers of CFU/g observed between dose escalation and de-escalation were significant for the hVISA strain, with the de-escalation simulation exhibiting a better killing effect than the escalation simulation (P < 0.024). Although our results need to be carefully considered, the use of high-dose daptomycin up front demonstrated the most efficient activity against the tested isolates. Different therapeutic scenarios including isolates with higher MICs and prolonged drug exposures are warranted to better understand the outcomes of escalation and de-escalation strategies.We thank Debbie Goff and Preeti Pancholi from the Ohio State Medical Center for kindly providing isolate B010-01. This study was funded by a research grant from Cubist Pharmaceuticals. M.J.R. has received grant support, has served as a consultant, or has participated as a speaker for Astellas, Cerexa, Cubist, Forest, Johnson & Johnson, Pfizer, Targanta and Theravance. C.V. and M.E.S. have no potential conflicts to declare

Evaluation of Telavancin Activity versus Daptomycin and Vancomycin against Daptomycin-Nonsusceptible Staphylococcus aureus in an In Vitro Pharmacokinetic/Pharmacodynamic Model

Daptomycin-nonsusceptible (DNS) Staphylococcus aureus strains have been reported over the last several years. Telavancin is a lipoglycopeptide with a dual mechanism of action, as it inhibits peptidoglycan polymerization/cross-linking and disrupts the membrane potential. Three clinical DNS S. aureus strains, CB1814, R6212, and SA-684, were evaluated in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model with simulated endocardial vegetations (starting inoculum, 108.5 CFU/g) for 120 h. Simulated regimens included telavancin at 10 mg/kg every 24 h (q24h; peak, 87.5 mg/liter; t1/2, 7.5 h), daptomycin at 6 mg/kg q24h (peak, 95.7 mg/liter; t1/2, 8 h), and vancomycin at 1 g q12h (peak, 30 mg/liter; t1/2, 6 h). Differences in CFU/g between regimens at 24 through 120 h were evaluated by analysis of variance with a Tukey's post hoc test. Bactericidal activity was defined as a ≥3-log10 CFU/g decrease in colony count from the initial inoculum. MIC values were 1, 0.25, and 0.5 mg/liter (telavancin), 4, 2, and 2 mg/liter (daptomycin), and 2, 2, and 2 mg/liter (vancomycin) for CB1814, R6212, and SA-684, respectively. Telavancin displayed bactericidal activities against R6212 (32 to 120 h; −4.31 log10 CFU/g), SA-684 (56 to 120 h; −3.06 log10 CFU/g), and CB1814 (48 to 120 h; −4.9 log10 CFU/g). Daptomycin displayed initial bactericidal activity followed by regrowth with all three strains. Vancomycin did not exhibit sustained bactericidal activity against any strain. At 120 h, telavancin was significantly better at reducing colony counts than vancomycin against all three tested strains and better than daptomycin against CB1814 (P < 0.05). Telavancin displayed bactericidal activity in vitro against DNS S. aureus isolates.This study was funded by a research grant from Astellas, Deerfield, IL. M.J.R. has received grant support, has served as a consultant, or has participated as a speaker for Astellas, Cerexa, Cubist, Forest, Pfizer, and Theravance. C.V. and M.E.S. have no conflicts to declare

mHealth: providing a mindfulness app for women with chronic pelvic pain in gynaecology outpatient clinics: qualitative data analysis of user experience and lessons learnt

OBJECTIVES: To determine whether a pre-existing smartphone app to teach mindfulness meditation is acceptable to women with chronic pelvic pain (CPP) and can be integrated into clinical practice within the National Health Service (NHS) CPP pathways, and to inform the design of a potential randomised clinical trial. DESIGN: A prestudy patient and public involvement (PPI) group to collect feedback on the acceptability of the existing app and study design was followed by a three-arm randomised feasibility trial. In addition, we undertook interviews and focus groups with patients and staff to explore app usability and acceptability. We also obtained participant comments on the research process, such as acceptability of the study questionnaires. SETTING: Two gynaecology clinics within Barts Health NHS, London, UK. PARTICIPANTS: Patients with CPP lasting ≥6 months with access to smartphone or personal computer and understanding of basic English. INTERVENTION: The intervention was mindfulness meditation content plus additional pain module delivered by a smartphone app. Active controls received muscle relaxation content from the same app. Passive (waiting list) controls received usual care. MAIN OUTCOME MEASURES: Themes on user feedback, app usability and integration, and reasons for using/not using the app. RESULTS: The use of the app was low in both active groups. Patients in the prestudy PPI group, all volunteers, were enthusiastic about the app (convenience, content, portability, flexibility, ease of use). Women contributing to the interview or focus group data (n=14), from a 'real world' clinic (some not regular app users), were less positive, citing as barriers lack of opportunities/motivation to use the app and lack of familiarity and capabilities with technology. Staff (n=7) were concerned about the potential need for extra support for them and for the patients, and considered the app needed organisational backing and peer acceptance. CONCLUSION: The opinions of prestudy PPI volunteers meeting in their private time may not represent those of patients recruited at a routine clinic appointment. It may be more successful to codesign/codevelop an app with typical users than to adapt existing apps for use in real-world clinical populations. TRIAL REGISTRATION NUMBER: ISRCTN10925965

Novel Daptomycin Combinations against Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus in an In Vitro Model of Simulated Endocardial Vegetations

Reduced susceptibility to daptomycin has been reported in patients with infections due to methicillin-resistant Staphylococcus aureus (MRSA). Although infections with daptomycin-nonsusceptible (DNS) MRSA are infrequent, optimal therapy of these strains has not been determined. We investigated the killing effects of novel antibiotic combinations with daptomycin (DAP) against two clinical DNS MRSA isolates (SA-684 and R6003) in a 72-h in vitro pharmacokinetic/pharmacodynamic (PK/PD) model with simulated endocardial vegetations (SEV). Simulated regimens included DAP at 6 mg/kg every 24 h (q24h) alone or in combination with trimethoprim-sulfamethoxazole (TMP/SMX) at 160/800 mg q12h, linezolid (LIN) at 600 mg q12h, cefepime (CEF) at 2 g q12h, and nafcillin (NAF) at 4 g q4h. Bactericidal activity was defined as a ≥3-log10 CFU/g kill. Differences in CFU/g were evaluated between 4 and 72 h by analysis of variance with the Bonferroni post hoc test. DAP MICs were 4 and 2 mg/liter for SA-684 and R6003, respectively. In the PK/PD model, DAP alone was slowly bactericidal, achieving a 3-log10 kill at 24 and 50 h for SA-684 and R6003, respectively. Against SA-684, DAP plus TMP/SMX, CEF, LIN, or NAF was bactericidal at 4, 4, 8, and 8 h, respectively, and maintained this activity for the 72-h study duration. DAP plus TMP/SMX or CEF exhibited superior killing than DAP alone against SA-684 between 4 and 72 h, and overall this was significant (P < 0.05). Against R6003, DAP plus TMP/SMX was bactericidal (8 h) and superior to DAP alone between 8 and 72 h (P < 0.001). The unique combination of DAP plus TMP/SMX was the most effective and rapidly bactericidal regimen against the two isolates tested and may provide a clinical option to treat DNS S. aureus infections.This work was not funded by any external support. M.J.R. has received grant support, consulted for, or provided lectures for Astellas, Cubist, Forrest, Ortho-McNeil, and Pfizer