Data_Sheet_1_Melanopsin DNA aptamers can regulate input signals of mammalian circadian rhythms by altering the phase of the molecular clock.pdf

DNA aptamers can bind specifically to biomolecules to modify their function, potentially making them ideal oligonucleotide therapeutics. Herein, we screened for DNA aptamer of melanopsin (OPN4), a blue-light photopigment in the retina, which plays a key role using light signals to reset the phase of circadian rhythms in the central clock. Firstly, 15 DNA aptamers of melanopsin (Melapts) were identified following eight rounds of Cell-SELEX using cells expressing melanopsin on the cell membrane. Subsequent functional analysis of each Melapt was performed in a fibroblast cell line stably expressing both Period2:ELuc and melanopsin by determining the degree to which they reset the phase of mammalian circadian rhythms in response to blue-light stimulation. Period2 rhythmic expression over a 24-h period was monitored in Period2:ELuc stable cell line fibroblasts expressing melanopsin. At subjective dawn, four Melapts were observed to advance phase by >1.5 h, while seven Melapts delayed phase by >2 h. Some Melapts caused a phase shift of approximately 2 h, even in the absence of photostimulation, presumably because Melapts can only partially affect input signaling for phase shift. Additionally, some Melaps were able to induce phase shifts in Per1::luc transgenic (Tg) mice, suggesting that these DNA aptamers may have the capacity to affect melanopsin in vivo. In summary, Melapts can successfully regulate the input signal and shifting phase (both phase advance and phase delay) of mammalian circadian rhythms in vitro and in vivo.</p

Novel Parallelized Electroporation by Electrostatic Manipulation of a Water-in-Oil Droplet as a Microreactor

<div><p>Electroporation is the most widely used transfection method for delivery of cell-impermeable molecules into cells. We developed a novel gene transfection method, water-in-oil (W/O) droplet electroporation, using dielectric oil and an aqueous droplet containing mammalian cells and transgene DNA. When a liquid droplet suspended between a pair of electrodes in dielectric oil is exposed to a DC electric field, the droplet moves between the pair of electrodes periodically and droplet deformation occurs under the intense DC electric field. During electrostatic manipulation of the droplet, the local intense electric field and instantaneous short circuit via the droplet due to droplet deformation facilitate gene transfection. This method has several advantages over conventional transfection techniques, including co-transfection of multiple transgene DNAs into even as few as 10³ cells, transfection into differentiated neural cells, and the capable establishment of stable cell lines. In addition, there have been improvements in W/O droplet electroporation electrodes for disposable 96-well plates making them suitable for concurrent performance without thermal loading by a DC electric field. This technique will lead to the development of cell transfection methods for novel regenerative medicine and gene therapy.</p></div

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Double and stable transfected cells by W/O droplet electroporation.

<p>(A), (B) Transgene plasmid DNAs of two kinds of fluorescent protein: green fluorescent protein (GFP) and Tag-RFP (red fluorescent protein), were successfully double transfected into HEK293 cells by W/O droplet electroporation for 3 minutes. Bright-field image and fluorescent GFP or RFP image of HEK293 cells (A) 5 days (71% of cells maintained GFP and 71% of cells maintained TagRFP signal) and (B) 8 days (41% of cells maintained GFP and 37% of cells maintained TagRFP signal) after W/O droplet electroporation. Scale bars, 30 μm. (C) Transgene plasmid DNAs of Venus and mCherry (red FP) were successfully double transfected into hippocampus primary neural cell lines by W/O droplet electroporation for 5 minutes. Scale bars, 30 μm. (D), (E) Bright-field image and fluorescent image of Venus and mCherry expression in human fibroblast cells (D) 2 days and (E) 10 days after W/O droplet electroporation. Scale bars, 30 μm.</p

Fibroblast, SCN neural cells and HEK293 cells transfected Venus plasmid by W/O droplet electroporation.

<p>(A) Bright field, fluorescence and merge images of fibroblast cells from a subject aged 81 years old one day after electroporation. (B) 7 days after W/O droplet electroporation. (C) Bright field, fluorescence and merge images of SCN neural cells three days after W/O droplet electroporation. SCN cells had been differentiated to neural cells by incubation at higher culture temperature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144254#pone.0144254.ref026" target="_blank">26</a>]. (D) Twelve days after W/O droplet electroporation. All scale bars (A-D), 30 μm. (E) Bright field, fluorescence and merge images of HEK293 cells colonies were picked up and cultured for one month after W/O droplet electroporation. (F) Bright field, fluorescence and merge images of HEK293 cells colonies were cultured though frozen stock for a total of more than two months after W/O droplet electroporation. scale bars (D-E), 100 μm.</p

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The droplet actuation device and cells transfected by the W/O droplet electroporation method.

<p>(A), (B) Two pieces of conductive tape were set parallel on a single cuvette or 8 wells in a line for 96-well plastic microwell plates. The droplet continued to bounce between the edges of the two electrodes. One was the ground electrode, and the other was the high-voltage electrode. Images of droplets bouncing between the anode and cathode in each well are shown. Many cells in droplets can be transfected simultaneously. (C) The upper row : Bright-field (BF), fluorescence, and merge images of HEK293 cells 24 hours after transfection by W/O droplet electroporation. Cells were examined at 24 hours after transfection to evaluate the expression of fluorescent protein (Venus). Scale bars, 100 μm. The lower row : BF, fluorescence, and merge images of HEK293 cells 24 hours after transfection by lipofection. (D) Variation of cell viability with time of droplet actuation determined by trypan blue staining. All experiments were performed at least twice.</p

Image of the parallel W/O droplet electroporation electrode for the 8-well string of disposable 96-well plates.

<p>(A) Conductive electrodes were produced and set on an 8-well string in 96-well microwell plates for W/O droplet electroporation. (B), (C) Venus plasmid was transfected into some HEK293 cells using 8-well W/O droplet electroporation electrodes (Nepa Gene) for 96-well plates at 1.8–2.2 kV. Venus fluorescence signals were observed after incubation in 5 of 8 wells for 4 and 7 days following electroporation. Scale bars, 20 μm.</p

Additional file 2: of A central-acting connexin inhibitor, INI-0602, prevents high-fat diet-induced feeding pattern disturbances and obesity in mice

Figure S2. The SFAs in HFDs disrupted feeding patterns by increasing light cycle intake. (a–c) Mice were fed one of two diets high in saturated fatty acids (SFAs); the HFD60 contained higher amounts of C16:0 and C18:0 (n = 6, black circles with solid line) than the HFD32 (n = 8, white squares with dashed line). After acclimation to FDAMS with NC, mice were fed the HFDs for 5 days. White and black bars on the X-axis correspond to the light and dark cycles, respectively. (a-b) Hourly caloric intake (1 kcal = 4.186 kJ) before (Pre) and after the diet switch from NC to (a) HFD60 or (b) HFD32. (c) The light cycle intake expressed as a percentage of the 24-h intake. Black circles with solid line: the HFD60 group; white squares with dashed line: the HFD32 group. Data are the means ± s.e.m. Statistical significance was determined with the Student’s paired t-test for comparisons to pre-diet values for each group, in c; *P < 0.05. Abbreviations: NC, normal chow; HFD, high-fat diet; FDAMS, feeding drinking, and activity monitoring system. (TIF 226 kb