Silk fibroin is increasingly emerging as an important biomaterial for tissue engineering applications. The ability to fluorescently image silk matrices under a microscope would be helpful in differentiating embedded labeled cells from background signal, critical for the study of silk-based engineered tissues. In this study, we fabricated a scaffold using freeze drying and confirmed its structure by X-ray diffraction and Fourier transform infrared spectroscopy. We then examined the fluorescence of the silk fibroin scaffold using confocal microscopy, both before and after cell seeding and fluorescent labeling. We subsequently investigated the fluorescent signature of the silk fibroin scaffold chemically. Fluorophore-labeled cells seeded into the scaffold showed the same fluorescent color as the scaffold itself when excited by the same wavelength of light. UV-Vis and fluorescence spectroscopy of a silk fibroin solution indicated absorption and emission maxima at 277 and 345 nm, respectively, which is a typical protein-derived signal. HPLC and GC-MS were used to detect quercetin and quercetin derivatives, without success. We therefore conclude that unlike silk cocoons, the fluorescent behavior of silk fibroin scaffolds does not derive from quercetin and its derivatives but from the intrinsic fluorescence of fibroin protein. We also find that the fluorescent signals deriving from a scaffold and from labeled cells embedded in it can be distinguished when the different optical channels are merged.

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We designed a study to induce differentiation of Oct4-GFP (expression of Green Fluorescent Protein of oct4) embryonic stem cells (ESCs) by embryoid body (EB) culture system into germ cells (GCs) using retinoic acid (RA) and evaluated the expression level of (Fkbp6, Mov10l1, 4930432K21Rik, and Tex13) in differentiated cells. The expression levels of four GC-related genes, Oct4, Mvh, Scp3, and Stra8, was determined by quantitative real-time polymerase chain reaction (q-RT-PCR). Immunostaining and flow cytometry were used as additional tests to confirm q-RT-PCR findings. A significant increase occurred in the expression of meiotic markers and specific genes, Fkbp6 (p = 0.00), Mov10l1 (p = 0.01), and Tex13 (p = 0.00) in ESCs treated with RA (+RA) compared with the controls (-RA). Oct4 expression was decreased in all studied groups. The expression levels of 4930432K21Rik, Mvh, Stra8, and Scp3 in the +RA group was higher than that of the -RA group. Flow cytometry analysis showed that mean number of Mvh-positive cells in the +RA group was greater as compared with ESCs, -RA and EB7 groups (p = 0.00). Downregulation of Oct4 as a pluripotency factor as well as the expression of meiosis markers, this hypothesis is raised that ESCs are differentiated by RA, and have been introduced into the zygote/pachytene of first meiosis as GC-like cells.

The nucleus accumbens shell (NAcSh) plays a role in appetitive and negative motivation with sex differences in responses. NAcSh and its laterality in metabolic and hormonal responses to chronic stress in female rats is evaluated via transient inactivation of this nucleus during stress induction. Animals in the stress groups received consecutive stress for four days and transient inactivation of NAcSh was performed by administrating lidocaine (0.2%) unilaterally or bilaterally in the nucleus for five minutes before electric foot shock induction. After stress termination, food and water intake, latency to eat, plasma glucose, corticosterone, estradiol and progesterone were measured in all groups. Results showed that stress increased food intake and blood glucose level, but there were no change in the latency to eat and the amount of water intake. The right side, the left side, and both sides of NAcSh may be dominant in latency to eat, food intake, and both water intake and plasma glucose level, respectively. Although chronic stress included no changes for corticosterone and progesterone, it increased estradiol level in plasma. Also, bilateral and right sides of NAcSh may have modulatory effects on stress in corticosterone and progesterone, respectively, HIGHLIGHTS  Chronic stress increase food intake and blood glucose significantly.  Chronic stress has no effect on delay to start eating and amount of water intake.  Right side and left side of NAcSh are dominant in delay and amount of food intake.  Stress increase estradiol level but has no effect on corticosterone and progesterone. 2 Sahraei, H.; et al.