Urology Program Directors' Perception of Pregnancy During Residency.

OBJECTIVE: To assess urology program directors' (PDs) perception of pregnancy during residency training. METHODS: A 30 question anonymous survey was sent to 142 urology PDs regarding their demographics, program information, institution policies, and self-reported opinions. Results were assessed via descriptive analysis. RESULTS: A total of 63 PDs responded with a response rate of 44%: 19% were female, 73% between 40 and 59 years of age, and 91% had children. A minority (17%) of programs had 40% or more female residents. 37% of PDs had never had a pregnant resident during their time as PD while 57% had 1 to 5 pregnant residents. On multivariate analysis, PDs age > 60 years or PD having their first child when > 30 years old were predictors for poor support of maternity leave. The majority of PDs felt their program was better/much better at being supportive toward maternity leave compared to other surgical specialties at their institution. Only 21% of PDs felt that taking maternity leave burdened other residents unfairly. Of respondents, 62% felt prepared/completely prepared to advise residents on pregnancy during residency. However, 91% of PDs affirmed it would be helpful to have formal policies in place regarding maternity/paternity leave. CONCLUSION: While the majority of PDs do not have a negative perception of pregnancy during residency, a small portion feels that pregnancy during residency is a burden on other residents. More than half of PDs feel prepared to discuss this issue with their residents. However, a large majority would find formal policies helpful.

Phase-Separated Liposomes Enhance the Efficiency of Macromolecular Delivery to the Cellular Cytoplasm.

INTRODUCTION: From viruses to organelles, fusion of biological membranes is used by diverse biological systems to deliver macromolecules across membrane barriers. Membrane fusion is also a potentially efficient mechanism for the delivery of macromolecular therapeutics to the cellular cytoplasm. However, a key shortcoming of existing fusogenic liposomal systems is that they are inefficient, requiring a high concentration of fusion-promoting lipids in order to cross cellular membrane barriers. OBJECTIVES: Toward addressing this limitation, our experiments explore the extent to which membrane fusion can be amplified by using the process of lipid membrane phase separation to concentrate fusion-promoting lipids within distinct regions of the membrane surface. METHODS: We used confocal fluorescence microscopy to investigate the integration of fusion-promoting lipids into a ternary lipid membrane system that separated into liquid-ordered and liquid-disordered membrane phases. Additionally, we quantified the impact of membrane phase separation on the efficiency with which liposomes transferred lipids and encapsulated macromolecules to cells, using a combination of confocal fluorescence imaging and flow cytometry. RESULTS: Here we report that concentrating fusion-promoting lipids within phase-separated lipid domains on the surfaces of liposomes significantly increases the efficiency of liposome fusion with model membranes and cells. In particular, membrane phase separation enhanced the delivery of lipids and model macromolecules to the cytoplasm of tumor cells by at least 4-fold in comparison to homogenous liposomes. CONCLUSIONS: Our findings demonstrate that phase separation can enhance membrane fusion by locally concentrating fusion-promoting lipids on the surface of liposomes. This work represents the first application of lipid membrane phase separation in the design of biomaterials-based delivery systems. Additionally, these results lay the ground work for developing fusogenic liposomes that are triggered by physical and molecular cues associated with target cells.

Erratum to: Phase-Separated Liposomes Enhance the Efficiency of Macromolecular Delivery to the Cellular Cytoplasm.

[This corrects the article DOI: 10.1007/s12195-017-0489-4.].

Steric Pressure among Membrane-Bound Polymers Opposes Lipid Phase Separation.

Lipid rafts are thought to be key organizers of membrane-protein complexes in cells. Many proteins that interact with rafts have bulky polymeric components such as intrinsically disordered protein domains and polysaccharide chains. Therefore, understanding the interaction between membrane domains and membrane-bound polymers provides insights into the roles rafts play in cells. Multiple studies have demonstrated that high concentrations of membrane-bound polymeric domains create significant lateral steric pressure at membrane surfaces. Furthermore, our recent work has shown that lateral steric pressure at membrane surfaces opposes the assembly of membrane domains. Building on these findings, here we report that membrane-bound polymers are potent suppressors of membrane phase separation, which can destabilize lipid domains with substantially greater efficiency than globular domains such as membrane-bound proteins. Specifically, we created giant vesicles with a ternary lipid composition, which separated into coexisting liquid ordered and disordered phases. Lipids with saturated tails and poly(ethylene glycol) (PEG) chains conjugated to their head groups were included at increasing molar concentrations. When these lipids were sparse on the membrane surface they partitioned to the liquid ordered phase. However, as they became more concentrated, the fraction of GUVs that were phase-separated decreased dramatically, ultimately yielding a population of homogeneous membrane vesicles. Experiments and physical modeling using compositions of increasing PEG molecular weight and lipid miscibility phase transition temperature demonstrate that longer polymers are the most efficient suppressors of membrane phase separation when the energetic barrier to lipid mixing is low. In contrast, as the miscibility transition temperature increases, longer polymers are more readily driven out of domains by the increased steric pressure. Therefore, the concentration of shorter polymers required to suppress phase separation decreases relative to longer polymers. Collectively, our results demonstrate that crowded, membrane-bound polymers are highly efficient suppressors of phase separation and suggest that the ability of lipid domains to resist steric pressure depends on both their lipid composition and the size and concentration of the membrane-bound polymers they incorporate.

Combination of 13 cis-retinoic acid and tolfenamic acid induces apoptosis and effectively inhibits high-risk neuroblastoma cell proliferation.

Chemotherapeutic regimens used for the treatment of Neuroblastoma (NB) cause long-term side effects in pediatric patients. NB arises in immature sympathetic nerve cells and primarily affects infants and children. A high rate of relapse in high-risk neuroblastoma (HRNB) necessitates the development of alternative strategies for effective treatment. This study investigated the efficacy of a small molecule, tolfenamic acid (TA), for enhancing the anti-proliferative effect of 13 cis-retinoic acid (RA) in HRNB cell lines. LA1-55n and SH-SY5Y cells were treated with TA (30µM) or RA (20µM) or both (optimized doses, derived from dose curves) for 48h and tested the effect on cell viability, apoptosis and selected molecular markers (Sp1, survivin, AKT and ERK1/2). Cell viability and caspase activity were measured using the CellTiter-Glo and Caspase-Glo kits. The apoptotic cell population was determined by flow cytometry with Annexin-V staining. The expression of Sp1, survivin, AKT, ERK1/2 and c-PARP was evaluated by Western blots. The combination therapy of TA and RA resulted in significant inhibition of cell viability (p<0.0001) when compared to individual agents. The anti-proliferative effect is accompanied by a decrease in Sp1 and survivin expression and an increase in apoptotic markers, Annexin-V positive cells, caspase 3/7 activity and c-PARP levels. Notably, TA+RA combination also caused down regulation of AKT and ERK1/2 suggesting a distinct impact on survival and proliferation pathways via signaling cascades. This study demonstrates that the TA mediated inhibition of Sp1 in combination with RA provides a novel therapeutic strategy for the effective treatment of HRNB in children.