PCI-24781 can improve in vitro and in vivo developmental capacity of pig somatic cell nuclear transfer embryos

Objective

To examine the effect of PCI-24781 (abexinostat) on the blastocyst formation rate in pig somatic cell nuclear transferred (SCNT) embryos and acetylation levels of the histone H3 lysine 9 and histone H4 lysine 12.

Results

Treatment with 0.5 nM PCI-24781 for 6 h significantly improved the development of cloned embryos, in comparison to the control group (25.3 vs. 10.5 %, P < 0.05). Furthermore, PCI-24781 treatment led to elevated acetylation of H3K9 and H4K12. TUNEL assay and Hoechst 33342 staining revealed that the percentage of apoptotic cells in blastocysts was significantly lower in PCI-24781-treated SCNT embryos than in untreated embryos. Also, PCI-24781-treated embryos were transferred into three surrogate sows, one of whom became pregnant and two fetuses developed.

Conclusion

PCI-24781 improves nuclear reprogramming and the developmental potential of pig SCNT embryos.

     

Generation of ΔF508-CFTR T84 cell lines by CRISPR/Cas9-mediated genome editing

Objectives: To provide a simple method to make a stable ΔF508-CFTR-expressing T84 cell line that can be used as an efficient screening model system for ΔF508-CFTR rescue. Results: CFTR knockout cell lines were generated by Cas9 with a single-guide RNA (sgRNA) targeting exon 1 of the CFTR genome, which produced indels that abolished CFTR protein expressions. Next, stable ΔF508-CFTR expression was achieved by genome integration of ΔF508-CFTR via the lentivirus infection system. Finally, we showed functional rescue of ΔF508-CFTR not only by growing the cells at a low temperature, but also incubating with VX-809, a ΔF508-CFTR corrector, in the established T84 cells expressing ΔF508-CFTR. Conclusions: This cell system provides an appropriate screening platform for rescue of ΔF508-CFTR, especially related to protein folding, escaped from endoplasmic-reticulum-associated protein degradation, and membrane transport.     

Production of δ-decalactone from linoleic acid via 13-hydroxy-9(Z)-octadecenoic acid intermediate by one-pot reaction using linoleate 13-hydratase and whole <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cells

Objective

To produce δ-decalactone from linoleic acid by one-pot reaction using linoleate 13-hydratase with supplementation with whole Yarrowia lipolytica cells.

Results

Whole Y. lipolytica cells at 25 g l^?1 produced1.9 g l^?1 δ-decalactone from 7.5 g 13-hydroxy-9(Z)-octadecenoic acid l^?1 at pH 7.5 and 30 °C for 21 h. Linoleate 13-hydratase from Lactobacillus acidophilus at 3.5 g l^?1 with supplementation with 25 g Y. lipolytica cells l^?1 in one pot at 3 h produced 1.9 g l^?1 δ-decalactone from 10 g linoleic acid l^?1 via 13-hydroxy-9(Z)-octadecenoic acid intermediate at pH 7.5 and 30°C after 18 h, with a molar conversion yield of 31 % and productivity of 106 mg l^?1 h^?1.

Conclusion

To the best of our knowledge, this is the first production of δ-decalactone using unsaturated fatty acid.

     

Aging exo-enzymes can create temporally shifting,temperature-dependent resource landscapes for microbes

The rate at which catalytic capacity of microbial exo-enzymes degrades post-exudation will influence the time during which return on microbes’ investment in exo-enzyme production can be realized. Further, if exo-enzyme degradation rates vary across exo-enzymes, microbial investment returns may vary by element across time. We quantify how aging of two soil organic matter (SOM)-decaying enzymes (β-D-cellobioside, BGase; and N-acetyl-β-D-glucosaminide, NAGase) influences enzyme-substrate V _max at multiple temperatures (5, 15, 25 °C), and compute how enzyme age influences relative availabilities of C and N. Both BGase and NAGase exhibited similar, exponential declines in catalytic rate with age at 25 °C (0.22 ± 0.02 and 0.36 ± 0.14 d^?1, respectively). At 15 °C, NAGase exhibited exponential declines in catalytic rates with age (0.79 ± 0.31 d^?1), but BGase exhibited no decline. Neither enzyme exhibited a decline in catalytic rate over 72 h at 5 °C. At 15 °C, the amount of C liberated from cellulose and chitin analogues relative to N increased, on average, by more than one order of magnitude. The ratio of C:N liberated from the two substrates remained constant across enzyme age at 25 and 5 °C, but for different reasons: no differences in decay rate across enzymes at 25 °C, and no observed decay at 5 °C. Thus, temperature-dependent decreases of catalytic activity over time may influence microbial resource allocation strategies and rates of SOM decomposition. Because the enzyme decay rates we observed differ considerably from values assumed in most models, such assumptions should be revisited when parameterizing microbial process models.