Theoretical insights into excited-state intramolecular and multiple intermolecular hydrogen bonds in 2-(2-Hydroxy-phenyl)-4(3H)-quinazolinone.

The photophysical properties and photochemistry reactions of 2-(2-Hydroxy-phenyl)-4(3H)-quinazolinone (HPQ) system in different solutions are studied by using density functional theory (DFT) and time-dependent density functional theory (TDDFT) methods. Our theoretical investigation explores that an ultrafast barrier-free excited state intramolecular proton transfer (ESIPT) process occurs and the configuration twisting is found in the electronic excited state. In the polar protic methanol solution, the hydrogen-bonded complex composed by HPQ and two methanol molecules (HPQ-2M) could exist stably in the ground state. Upon photoexcitation the isolated HPQ is initially excited to the first excited state, while the HPQ-2M system is firstly excited to the S3 state and undergoes internal conversion (IC) to the S1 state. The intermolecular hydrogen bonds are strengthened in the excited state. The simulated electronic spectra agree well with the experimental results. The strengthening of the intermolecular hydrogen bonds is also confirmed by the calculated vibrational spectra. In addition, the intramolecular charge transfer happens in both HPQ and HPQ-2M systems from the frontier molecular orbital analysis.

A three-dimensional culture system using alginate hydrogel prolongs hatched cattle embryo development in vitro.

No successful method exists to maintain the three-dimensional architecture of hatched embryos in vitro. Alginate, a linear polysaccharide derived from brown algae, has characteristics that make it an ideal material as a three-dimensional (3D) extracellular matrix for in vitro cell, tissue, or embryo culture. In this study, alginate hydrogel was used for IVC of posthatched bovine embryos to observe their development under the 3D system. In vitro-fertilized and parthenogenetically activated posthatched bovine blastocysts were cultured in an alginate encapsulation culture system (AECS), an alginate overlay culture system (AOCS), or control culture system. After 18 days of culture, the survival rate of embryos cultured in AECS was higher than that in the control group (P < 0.05), and the embryos were expanded and elongated in AECS with the maximal length of 1.125 mm. When the AECS shrinking embryos were taken out of the alginate beads on Day 18 and cultured in the normal culture system, 9.09% of them attached to the bottoms of the plastic wells and grew rapidly, with the largest area of an attached embryo being 66.00 mm(2) on Day 32. The embryos cultured in AOCS developed monovesicular or multivesicular morphologies. Total cell number of the embryos cultured in AECS on Day 19 was significantly higher than that of embryos on Day 8. Additionally, AECS and AOCS supported differentiation of the embryonic cells. Binuclear cells were visible in Day-26 adherent embryos, and the messenger RNA expression patterns of Cdx2 and Oct4 in AOCS-cultured embryos were similar to those in vivo embryos, whereas IFNT and ISG15 messenger RNA were still expressed in Day-26 and Day-32 prolong-cultured embryos. In conclusion, AECS and AOCS did support cell proliferation, elongation, and differentiation of hatched bovine embryos during prolonged IVC. The culture system will be useful to further investigate the molecular mechanisms controlling ruminant embryo elongation and implantation.

Establishment and cryopreservation of a skin fibroblast cell line derived from Yunnan semi-fine wool sheep in the presence of synthetic ice blocker.

In this study, the fibroblasts cell line derived from ear marginal tissue of Yunnan semi-fine wool sheep was successfully established using the primary explants technique and cryopreservation technology. Additionally, the protective effect of synthetic ice blocker (SIB) including 1, 3-cyclohexanediol (1, 3-CHD) and 1, 4-cyclohexanediol (1, 4-CHD) on frozen fibroblast cells was also assessed and compared. Propidium iodide (PI) was used to stain the dead cells following cryopreservation and thawing. The results showed that compared with Medium 199 (M199) and Dulbecco's modified Eagle's medium : Nutrient Mixture F-12 (1 : 1) Mixture (DMEM/F12), Dulbecco's modified Eagle's medium (DMEM) may be more suitable for the primary culture of fibroblast cells of Yunnan semi-fine wool sheep. The growth curve of cells is a typical "S" type. After subculture for four days, the cells entered the plateau phase and began to degenerate. Biological analysis showed that the population doubling time (PDT) for subculturing fibroblast cells was approximately 26h. The Karyotyping data indicated that the percentage of fibroblast cells with normal chromosome number 2n = 54 was over 90% following subculture for 10 passages. Moreover, the tests for bacteria, fungi, viruses and mycoplasma were negative. After serial subculture for 5 generations, the fibroblast cells were cryopreserved in the presence or absence of 1, 3-CHD or 1, 4-CHD. The data indicated that with increase of the synthetic ice blocker concentrations, the viability of frozen-thawed fibroblast cells was firstly increased and then decreased. When the concentration of 1, 3-CHD or 1, 4-CHD was 50 mM, the viable percentage of frozen-thawed fibroblast cells was 91.93% +/- 2.24% and 94.13% +/- 0.55% respectively and significantly higher than that of the cells frozen in the absence of synthetic ice blockers (88.10% +/- 1.49%, P < 0.05). In conclusion, the skin fibroblast cell line of Yunnan semi-fine wool sheep was firstly established in this study. Additionally, the presence of synthetic ice blocker can increase the viability of frozen-thawed sheep fibroblast cell line.

The effects of trehalose and sucrose on frozen spermatozoa of Yunnan semi-fine wool sheep during a non-mating season.

To improve the quality of frozen spermatozoa of Yunnan semi-fine wool sheep, the protective effect of trehalose and sucrose on frozen ram spermatozoa during a non-mating season was evaluated and compared in this study. Briefly, following collection by electric stimulation, equilibration at degree C following dilution with the freezing extender, and pre-freezing in liquid nitrogen vapor, the ram spermatozoa were frozen in liquid nitrogen. After thawing, viability, motility, acrosome status, membrane integrity, and phosphatidylserine (PS) distribution was determined using a computer-assisted spermatozoa analysis system and flow cytometry. The data indicated disaccharide can improve the quality of frozen ram spermatozoa. With a trehalose concentration of 100mM, the post-thaw viability and motility (80.56 +/- 6.89% and 46.07 +/- 5.84 %) of ram spermatozoa were significantly more than those of ram spermatozoa frozen with no disaccharide (65.46 +/- 18.96 % and 34.62 +/- 9.32%, P<0.05). However, the effect of sucrose on the viability, motility, and moving velocity of ram spermatozoa was similar to that of the control group (p > 0.05). Compared with sucrose, trehalose can significantly increase the motility of frozen ram spermatozoa (p<0.05). In addition, addition of trehalose or sucrose can efficiently protect the acrosome of frozen spermatozoa. Moreover, when the concentration of trehalose or sucrose was 100mM, the protective effect of trehalose or sucrose on the membrane integrity and PS distribution was significantly higher than that of the control group (p>0.05). However, the protective effect of trehalose on viability, moving velocity, acrosome status, membrane integrity, and PS distribution of frozen ram spermatozoa was similar to that of sucrose (p>0.05). In conclusion, the protective effect of trehalose on frozen sheep spermatozoa is superior to that of sucrose. Addition of 100mM of trehalose in the freezing extenders can improve the post-thaw quality of ram spermatozoa with respect to viability, motility, and linear velocity. Moreover, presence of disaccharide can protect acrosome and membrane of frozen sheep spermatozoa.