New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators

It had been known for decades that primordial follicles in mammalian ovaries are assembled with definite numbers and represent the ovarian reserve throughout the reproductive life. Intra-oocyte PI3K/mTOR pathways have been indicated to play a central role on the activation of primordial follicles. Genetic modified mouse models with chronic activation of PI3K/mTOR signals in primordial oocytes showed premature activation of all primordial follicles and eventually their exhaustion. On the other hand, this may suggest that, unlike chronic activation of PI3K/mTOR, its acute activation in infertility would activate primordial follicles, permitting fertility during the treatment. Previously, PI3K stimulators were reported as a temporary measure to accelerate primordial follicle activation and follicular development in both mouse and human, and were applied in the treatment of infertility in premature ovarian failure (POF) patients. To address whether mTOR stimulators could play similar role in the process, we transiently treated neonatal and aged mouse ovaries with mTOR stimulators-phosphatidic acid (PA) and propranolol. Our results demonstrated the stimulators increased activation of primordial follicles and the production of progeny. Human ovarian cortex cubes were also treated with mTOR or/and PI3K stimulators in vitro. When they were used separately, both of them showed similar promotive effects on primordial follicles. Surprisingly, after joint-treatment with the 2 kinds of stimulators together, synergistic effects on follicular development were observed. Based on increased efficiency of follicular activation in humans, here we propose in vitro transient treatment with mTOR and PI3K stimulators as an optimized protocol for the application in different clinical conditions with limited follicle reserve.     

Effect of Bacillus mesonae H20-5 Treatment on Rhizospheric Bacterial Community of Tomato Plants under Salinity Stress

Plant growth-promoting bacteria improve plant growth under abiotic stress conditions. However, their effects on microbial succession in the rhizosphere are poorly understood. In this study, the inoculants of Bacillus mesonae strain H20-5 were administered to tomato plants grown in soils with different salinity levels (EC of 2, 4, and 6 dS/m). The bacterial communities in the bulk and rhizosphere soils were examined 14 days after H20-5 treatment using Illumina MiSeq sequencing of the bacterial 16S rRNA gene. Although the abundance of H20-5 rapidly decreased in the bulk and rhizosphere soils, a shift in the bacterial community was observed following H20-5 treatment. The variation in bacterial communities due to H20-5 treatment was higher in the rhizosphere than in the bulk soils. Additionally, the bacterial species richness and diversity were greater in the H20-5 treated rhizosphere than in the control. The composition and structure of the bacterial communities varied with soil salinity levels, and those in the H20-5 treated rhizosphere soil were clustered. The members of Actinobacteria genera, including Kineosporia, Virgisporangium, Actinoplanes, Gaiella, Blastococcus, and Solirubrobacter, were enriched in the H20-5 treated rhizosphere soils. The microbial co-occurrence network of the bacterial community in the H20-5 treated rhizosphere soils had more modules and keystone taxa compared to the control. These findings revealed that the strain H20-5 induced systemic tolerance in tomato plants and influenced the diversity, composition, structure, and network of bacterial communities. The bacterial community in the H20-5 treated rhizosphere soils also appeared to be relatively stable to soil salinity changes.     

Protein kinase C can inhibit TRPC3 channels indirectly via stimulating protein kinase G

There are two known phosphorylation-mediated inactivation mechanisms for TRPC3 channels. Protein kinase G (PKG) inactivates TRPC3 by direct phosphorylation on Thr-11 and Ser-263 of the TRPC3 proteins, and protein kinase C (PKC) inactivates TRPC3 by phosphorylation on Ser-712. In the present study, we explored the relationship between these two inactivation mechanisms of TRPC3. HEK cells were first stably transfected with a PKG-expressing construct and then transiently transfected with a TRPC3-expressing construct. Addition of 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analog of diacylglycerol (DAG), elicited a TRPC3-mediated [Ca2+]i rise in these cells. This OAG-induced rise in [Ca2+]i could be inhibited by phorbol 12-myristate 13-acetate (PMA), an agonist for PKC, in a dose-dependent manner. Importantly, point mutations at two PKG phosphorylation sites (T11A-S263Q) of TRPC3 markedly reduced the PMA inhibition. Furthermore, inhibition of PKG activity by KT5823 (1 microM) or H8 (10 microM) greatly reduced the PMA inhibition of TRPC3. These data strongly suggest that the inhibitory action of PKC on TRPC3 is partly mediated through PKG in these PKG-overexpressing cells. The importance of this scheme was also tested in vascular endothelial cells, in which PKG plays a pivotal functional role. In these cells, OAG-induced [Ca2+]i rise was inhibited by PMA, which activates PKC, and by 8-BrcGMP and S-nitroso-N-acetylpenicillamine (SNAP), both of which activate PKG. Importantly, the PMA inhibition on OAG-induced [Ca2+]i rise was significantly reduced by PKG inhibitor KT5823 (1 microM) or DT-3 (500 nM), suggesting an important role of PKG in the PMA-induced inhibition of TRPC channels in native endothelial cells.     

The first complete plastome sequence of the basal asterid family Styracaceae (Ericales) reveals a large inversion

Plastome sequences are rich sources of information for resolving difficult phylogenetic relationships and provide genomic data for conservation studies. Here, the complete plastome sequence of Alniphyllum eberhardtii Guillaumin is reported, representing the first plastome of the basal asterid family Styracaceae (Ericales). The plastome is 155,384 bp in length and contains 79 protein-coding genes, 30 tRNA genes and 4 rRNA genes, totaling 113 unique genes with 19 genes in the inverted repeat region. Unusual features of the plastome include the presence a large 20-kb inversion in the Large Single-Copy region, the pseudogenization of the accD gene, and the loss of the second intron from clpP. The 20-kb inversion includes 14 genes and has not been previously reported in other Ericales plastomes. Thirty-nine plastid simple sequence repeats (SSRs) that may provide genetic resources for the conservation of this economically import timber plant are characterized. Phylogenetic results inferred from ML and MP analyses of 66 plastid genes and 26 taxa reveal that the Styracaceae are sister to a clade including Actinidiaceae and Ericaceae and suggest that complete plastomes are likely to be very helpful in resolving the basal relationships among Ericales families, which have resisted resolution in smaller phylogenetic data sets.     

Reduction of chronic graft injury with a new HTK-based preservation solution in a murine heart transplantation model

Objective

Aim of this study was to evaluate a new histidine-tryptophan-ketoglutarate (HTK)-based preservation solution on chronic isograft injury in comparison to traditional HTK solution.

Methods

Hearts of C57BL/6J (H-2b) mice were stored for 15 h in 0–4 °C cold preservation solution and then transplanted heterotopically into C57BL/6J (H-2b) mice. Three groups were evaluated: HTK, the base solution of a new preservation solution and hearts without cold ischemia (control). Time to restoration of heartbeat was measured (re-beating time). Strength of the heartbeat was palpated daily and scored on a 4-level scale (palpation score). Animals were sacrificed after 60 days of observation (24 h for TGF-β expression). The transplanted hearts were evaluated histologically for myocardial damage, vasculopathy and interstitial fibrosis. TGF-β expression was assessed immunohistologically. All investigators were blinded to the groups. ANOVA and LSD post hoc test were used for statistical analysis.

Results

The re-beating time was significantly shorter in hearts stored in the new solution (10.3 ± 2.6 min vs. HTK 14.2 ± 4.1 min; p < 0.05). The palpation score was significantly higher in hearts stored in the new solution (2.3 ± 0.4 vs. HTK 1.6 ± 0.5; p < 0.01). Hearts stored in the new solution showed a lower myocardial injury score (1.8 ± 0.2 vs. HTK 2.2 ± 0.7), less interstitial fibrosis (4.8 ± 1.9% vs. HTK 8.5 ± 3.8%, p < 0.05), less vasculopathy (14.7 ± 6.9% vs. 22.0 ± 23.2%; p = 0.06) and lower TGF-β1-expression (6.6 ± 1.4% vs. HTK 12.0 ± 4.6%).

Conclusion

The new HTK-based solution reduces the chronic isograft injury. This protective effect is likely achieved through several modifications and supplements into the new solution like N-acetyl-l-histidine, glycine, alanine, arginine and sucrose.