Reconstituting ring-rafts in bud-mimicking topography of model membranes.

During vesicular trafficking and release of enveloped viruses, the budding and fission processes dynamically remodel the donor cell membrane in a protein- or a lipid-mediated manner. In all cases, in addition to the generation or relief of the curvature stress, the buds recruit specific lipids and proteins from the donor membrane through restricted diffusion for the development of a ring-type raft domain of closed topology. Here, by reconstituting the bud topography in a model membrane, we demonstrate the preferential localization of cholesterol- and sphingomyelin-enriched microdomains in the collar band of the bud-neck interfaced with the donor membrane. The geometrical approach to the recapitulation of the dynamic membrane reorganization, resulting from the local radii of curvatures from nanometre-to-micrometre scales, offers important clues for understanding the active roles of the bud topography in the sorting and migration machinery of key signalling proteins involved in membrane budding

Fabrication of pyramidal probes with various periodic patterns and a single nanopore

The nanometer-scale patterned pyramidal probe with an electron beam-induced nanopore on the pyramid apex is an excellent candidate for an optical biosensor. The nanoapertures surrounded with various periodic groove patterns on the pyramid sides were fabricated using a focused ion beam technique, where the optical characteristics of the fabricated apertures with rectangular, circular, and elliptical groove patterns were investigated. The elliptical groove patterns on the pyramid were designed to maintain an identical distance between the grooves and the apex for the surface waves and, among the three patterns, the authors observed the highest optical transmission from the elliptically patterned pyramidal probe. A 103-fold increase of the transmitted optical intensity was observed after patterning with elliptical grooves, even without an aperture on the pyramid apex. The nanopore on the apex of the pyramid was fabricated using electron beam irradiation and was optically characterized

Clinical and Pathological Characteristics of Four Korean Patients with Limb-Girdle Muscular Dystrophy type 2B

Limb-girdle muscular dystrophy type 2B (LGMD2B), a subtype of autosomal recessive limb-girdle muscular dystrophy (ARLGMD), is characterized by a relatively late onset and slow progressive course. LGMD2B is known to be caused by the loss of the dysferlin protein at sarcolemma in muscle fibers. In this study, the clinical and pathological characteristics of Korean LGMD2B patients were investigated. Seventeen patients with ARLGMD underwent muscle biopsy and the histochemical examination was performed. For the immunocytochemistry, a set of antibodies against dystrophin, α, β, γ, δ-sarcoglycans, dysferlin, caveolin-3, and β-dystroglycan was used. Four patients (24%) showed selective loss of immunoreactivity against dysferlin at the sarcolemma on the muscle specimens. Therefore, they were classified into the LGMD2B category. The age at the onset of disease ranged from 9 yr to 33 yr, and none of the patients was wheelchair bound at the neurological examination. The serum creatine kinase (CK) was high in all the patients (4010-5310 IU/L). The pathologic examination showed mild to moderate dystrophic features. These are the first Korean LGMD2B cases with a dysferlin deficiency confirmed by immunocytochemistry. The clinical, pathological, and immunocytochemical findings of the patients with LGMD2B in this study were in accordance with those of other previous reports

Manipulating nutrient composition of microalgal growth media to improve biomass yield and lipid content of Micractinium pusillum

Biodiesel production from microalgae depends on the algal biomass and lipid content. Both biomass production and lipid accumulation are limited by several factors in which nutrients play a key role. We investigated the influences of micronutrients on biomass, and lipid content of Micractinium pusillum GU732425 cultivated in bold basal media (BBM). The average dry biomass of microalgal strain in control medium reached 0.34 ± 0.01 g /L, while doubling (2X) the levels of Mn and Cu concentration increased the dry biomass to 0.38 ± 0.01 and 0.37 ± 0.02 g /L, respectively. M. pusillum cultivated in control medium had a biomass of 0.82 ± 0.05 g/L and a lipid productivity of 0.33 ± 0.02 g/L after 17 day cultivation. The alga cultivated in BBM with 4X Mn or 4X Cu produced more biomass (1.25 ± 0.01 or 1.28 ± 0.04 g dw/L) and lipid productivity (0.45±0.04 or 0.47±0.05 g/L), respectively. M. pusillum cultivated in different growth media had fatty acid compositions mainly comprising linoleic (49-54%), palmitic (24-29%), linolenic (16-22%), and oleic acids (2-5%). These results can be used to maximize the production of microalgal biomass and lipids in optimally designed  photobioreactors.Key words: Micractinium pusillum, biomass, lipid production, media composition, fatty acids, trace metals

Differences in youngest-old, middle-old, and oldest-old patients who visit the emergency department

Objective As aging progresses, clinical characteristics of elderly patients in the emergency department (ED) vary by age. We aimed to study differences among elderly patients in the ED by age group. Methods For 2 years, patients aged 65 and older were enrolled in the study and classified into three groups: youngest-old, ages 65 to 74 years; middle-old, 75 to 84 years; and oldest-old, ≥85 years. Participants’ sex, reason for ED visit, transfer from another hospital, results of treatment, type of admission, admission department and length of stay were recorded. Results During the study period, a total 64,287 patients visited the ED; 11,236 (17.5%) were aged 65 and older, of whom 14.4% were 85 and older. With increased age, the female ratio (51.5% vs. 54.9% vs. 69.1%, P<0.001), medical causes (79.5% vs. 81.3% vs. 81.7%, P=0.045), and admission rate (35.3% vs. 42.8% vs. 48.5%, P<0.001) increased. Admissions to internal medicine (57.5% vs. 59.3% vs. 64.7%, P<0.001) and orthopedic surgery (8.5% vs. 11.6% vs. 13.8%, P< 0.001) also increased. The ratio of admission to intensive care unit showed no statistical significance (P=0.545). Patients over age 85 years had longer stays in the ED (330.9 vs. 378.9 vs. 407.2 minutes, P<0.001), were discharged home less (84.4% vs. 78.9% vs. 71.5%, P<0.001), and died more frequently (6.3% vs. 10.4% vs. 13.0%, P<0.001). Conclusion With increased age, the proportion of female patients and medical causes increased. Rates of admission and death increased with age and older patients had longer ED and hospital stays

5-lipoxygenase mediates docosahexaenoyl ethanolamide and N-arachidonoyl-L-alanine-induced reactive oxygen species production and inhibition of proliferation of head and neck squamous cell carcinoma cells

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.Abstract Background Endocannabinoids have recently drawn attention as promising anti-cancer agents. We previously observed that anandamide (AEA), one of the representative endocannabinoids, effectively inhibited the proliferation of head and neck squamous cell carcinoma (HNSCC) cell lines in a receptor-independent manner. In this study, using HNSCC cell lines, we examined the anti-cancer effects and the mechanisms of action of docosahexaenoyl ethanolamide (DHEA) and N-arachidonoyl-L-alanine (NALA), which are polyunsaturated fatty acid (PUFA)-based ethanolamides like AEA. Methods and Results DHEA and NALA were found to effectively inhibit HNSCC cell proliferation. These anti-proliferative effects seemed to be mediated in a cannabinoid receptor-independent manner, since the antagonist of cannabinoid receptor-1 (CB1) and vanilloid receptor-1 (VR1), two endocannabinoid receptors, did not reverse the ability of DHEA and NALA to induce cell death. Instead, we observed an increase in reactive oxygen species (ROS) production and a decrease of phosphorylated Akt as a result of DHEA and NALA treatment. Antioxidants efficiently reversed the inhibition of cell proliferation and the decrease of phosphorylated Akt induced by DHEA and NALA; inhibition of 5-lipoxygenase (5-LO), which is expected to be involved in DHEA- and NALA-degradation pathway, also partially blocked the ability of DHEA and NALA to inhibit cell proliferation and phosphorylated Akt. Interestingly, ROS production as a result of DHEA and NALA treatment was decreased by inhibition of 5-LO. Conclusions From these findings, we suggest that ROS production induced by the 5-LO pathway mediates the anti-cancer effects of DHEA and NALA on HNSCC cells. Finally, our findings suggest the possibility of a new cancer-specific therapeutic strategy, which utilizes 5-LO activity rather than inhibiting it

Retinoid production using metabolically engineered Escherichia coli with a two-phase culture system

<p>Abstract</p> <p>Background</p> <p>Retinoids are lipophilic isoprenoids composed of a cyclic group and a linear chain with a hydrophilic end group. These compounds include retinol, retinal, retinoic acid, retinyl esters, and various derivatives of these structures. Retinoids are used as cosmetic agents and effective pharmaceuticals for skin diseases. Retinal, an immediate precursor of retinoids, is derived by β-carotene 15,15'-mono(di)oxygenase (BCM(D)O) from β-carotene, which is synthesized from the isoprenoid building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Retinoids are chemically unstable and biologically degraded via retinoic acid. Although extensive studies have been performed on the microbial production of carotenoids, retinoid production using microbial metabolic engineering has not been reported. Here, we report retinoid production using engineered <it>Escherichia coli </it>that express exogenous BCM(D)O and the mevalonate (MVA) pathway for the building blocks synthesis in combination with a two-phase culture system using a dodecane overlay.</p> <p>Results</p> <p>Among the BCM(D)O tested in <it>E. coli</it>, the synthetic retinoid synthesis protein (SR), based on bacteriorhodopsin-related protein-like homolog (Blh) of the uncultured marine bacteria 66A03, showed the highest β-carotene cleavage activity with no residual intracellular β-carotene. By introducing the exogenous MVA pathway, 8.7 mg/L of retinal was produced, which is 4-fold higher production than that of augmenting the MEP pathway (<it>dxs </it>overexpression). There was a large gap between retinal production and β-carotene consumption using the exogenous MVA pathway; therefore, the retinal derivatives were analyzed. The derivatives, except for retinoic acid, that formed were identified, and the levels of retinal, retinol, and retinyl acetate were measured. Amounts as high as 95 mg/L retinoids were obtained from engineered <it>E. coli </it>DH5α harboring the synthetic <it>SR </it>gene and the exogenous MVA pathway in addition to <it>dxs </it>overexpression, which were cultured at 29°C for 72 hours with 2YT medium containing 2.0% (w/v) glycerol as the main carbon source. However, a significant level of intracellular degradation of the retinoids was also observed in the culture. To prevent degradation of the intracellular retinoids through <it>in situ </it>extraction from the cells, a two-phase culture system with dodecane was used. The highest level of retinoid production (136 mg/L) was obtained after 72 hours with 5 mL of dodecane overlaid on a 5 mL culture.</p> <p>Conclusions</p> <p>In this study, we successfully produced 136 mg/L retinoids, which were composed of 67 mg/L retinal, 54 mg/L retinol, and 15 mg/L retinyl acetate, using a two-phase culture system with dodecane, which produced 68-fold more retinoids than the initial level of production (2.2 mg/L). Our results demonstrate the potential use of <it>E. coli </it>as a promising microbial cell factory for retinoid production.</p