Pseudo painting/air bubble technique for planar lipid bilayers.

BACKGROUND: A functional reconstitution of channel proteins in planar lipid bilayers is still very versatile to study structure/function correlates under well-defined conditions at the single protein level. NEW METHOD: In this study we present an improved planar lipid bilayer technique in which an air bubble is used for stabilizing unstable/leaky bilayers or for removing excess lipids. The bubble can also be used as a tool for reducing the number of channels in the bilayer with the goal of having only one active channel in the membrane. RESULTS: Stable planar lipid bilayers are formed within seconds to minutes. In the case of multiple channel insertion the air bubble can be used to reduce the number of channels within minutes. COMPARISON WITH EXISTING METHOD(S): The simple improvement of the classical folding technique guarantees a very fast creation of stable bilayers even with difficult phospholipids in a conventional vertical bilayer set-up; it requires no modifications of the existing set-up. CONCLUSIONS: This technique is very easy to handle and guarantees successful single channel recordings for any kind of planar lipid bilayer experiment.

Reprogramming of fibroblasts from older women with pelvic floor disorders alters cellular behavior associated with donor age.

We aimed to derive induced pluripotent stem cell (iPSC) lines from vaginal fibroblasts from older women with pelvic organ prolapse. We examined the effect of donor age on iPSCs and on the cells redifferentiated from these iPSCs. Vaginal fibroblasts were isolated from younger and older subjects for reprogramming. iPSCs were generated simultaneously using an excisable polycistronic lentiviral vector expressing Oct4, Klf4, Sox2, and cMyc. The pluripotent markers of iPSCs were confirmed by immunocytochemistry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Spectral karyotyping was performed. The ability of the iPSCs to differentiate into three germ layers was confirmed by embryoid body and teratoma formation. Senescence marker (p21, p53, and Bax) expressions were determined by qRT-PCR and Western blot. The iPSCs were redifferentiated to fibroblasts and were evaluated with senescence-associated ß-galactosidase (SA) activity and mitotic index using time-lapse dark-field microscopy. iPSCs derived from both the younger and older subjects expressed pluripotency markers and showed normal karyotype and positive teratoma assays. There was no significant difference in expression of senescence and apoptosis markers (p21, p53, and Bax) in iPSCs derived from the younger subject compared with the older subject. Furthermore, fibroblasts redifferentiated from these iPSCs did not differ in SA activity or mitotic index. We report successful derivation of iPSCs from women with pelvic organ prolapse. Older age did not interfere with successful reprogramming. Donor age differences were not observed in these iPSCs using standard senescence markers, and donor age did not appear to affect cell mitotic activity in fibroblasts redifferentiated from iPSCs.

Autologous replacement of the vocal fold: a new surgical approach for adduction-type spasmodic dysphonia.

Many surgical approaches have been developed for the treatment of adduction-type spasmodic dysphonia (SPD). We developed and performed a new type of surgical approach (autologous replacement of the vocal fold). Our new surgical technique increases the advantages and decreases the disadvantages of previous surgical procedures in three ways: (1) It has similar effects to the previous procedures in that it prevents contraction of the thyroarytenoid muscle. (2) It decreases vocal-fold tension, as in framework surgery. (3) It reduces glottal incompetence, as does fibrinogen-glue injection, but it is more suitable because it is autologous. Furthermore, it produces increases in the mass and volume of the vocal-fold body and is also safe because the replacement tissue is autologous. The short-term results appear encouraging in preventing spastic voice while also avoiding vocal-fold atrophy. Long-term follow up will be necessary to determine the actual efficacy. However, this is clearly a possible choice as a surgical approach for treating adduction-type SPD.