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.     

Diversity of the bacterial community in Myanmar traditional salted fish <Emphasis Type="Italic">yegyo ngapi</Emphasis>

The distribution and characterization of bacteria including lactic acid bacteria (LAB) in the traditional and popular salted fish yegyo ngapi in Myanmar were studied to clarify the contribution of these bacteria to the curing and ripening of this product. Samples of yegyo ngapi purchased from a market in Yangon were used. Most of the isolates obtained using de Man, Rogosa and Sharpe medium containing 10 % NaCl were identified as coccoid LAB on the basis of their basic phenotypic characteristics. From the results of 16S rRNA gene sequencing and PCR-restriction fragment length polymorphism analysis of this gene, most of the isolates were identified as the halophilic LAB Tetragenococcus muriaticus. Analyses of the 16S rRNA gene based on the clone library using DNA extracted from salted fish products were also performed. The results of these molecular-analysis-based techniques showed that spore-forming and non-spore-forming anaerobic bacteria including the genera Clostridium and Halanaerobium in addition to T. muriaticus were also frequently found in bacterial communities. These findings suggest that the anaerobic condition during curing and ripening resulted in bacterial communities composed of strictly anaerobic bacteria and halophilic LAB, and that these bacteria might also contribute to the manufacturing processes of this product. In addition, DNA sequences similar to that of Clostridium botulinum were found in the clone library analysis. Therefore, despite no reports of botulism poisoning from the region where the samples were taken, closer surveillance should be carried out from the viewpoint of food safety.     

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.     

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.     

The influence of cholesterol and lipid metabolism on host cell structure and hepatitis C virus replication.

The hepatitis C virus (HCV) replicates on a membrane protein complex composed of viral proteins, replicating RNA, and altered cellular membranes. Small-molecule inhibitors of cellular lipid-cholesterol metabolism such as 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 all show a negative effect on HCV replication. Perturbation of host cell lipid and cholesterol metabolism can disrupt replication complexes by altering membranous structures where replication occurs. Changes in cholesterol and (or) lipid composition can have a general effect on membrane structure. Alternatively, metabolic changes can exert a more subtle influence over replication complexes by altering localization of host proteins through alterations in lipid anchoring. Here, we use Huh-7 cells harboring subgenomic HCV replicons to demonstrate that 25-hydroxycholesterol, cerulenin, lovastatin, and GGTI-286 do not disrupt the membranous web where replication occurs, whereas cholesterol-depleting agents such as beta-cyclodextrin do. Cellular imaging suggests that the HCV RNA can remain associated with subcellular compartments connected with replication complexes in the presence of metabolic inhibitors. Therefore, at least 2 different molecular mechanisms are possible for the inhibition of HCV replication through the modulation of cellular lipid and cholesterol metabolism.     

Blastocysts derivation from somatic cell fusion with premature oocytes (prematuration somatic cell fusion)

This study was undertaken to investigate the development of immature oocytes after their fusion with male somatic cells expressing red fluorescence protein (RFP). RFP‐expressing cells were fused with immature oocytes, matured in vitro and then parthenogenetically activated. Somatic nuclei showed spindle formation, 1st polar body extrusion after in vitro maturation and protruded the 2nd polar body after parthenogenetic activation. RFP was expressed in the resultant embryos; two‐cell stage and blastocysts. Chromosomal analysis showed aneuploidy in 81.82% of the resulting blastocysts while 18.18% of the resulting blastocysts were diploid. Among eight RFP‐expressing blastocysts, Xist mRNAs was detected in six while Sry mRNA was detected in only one blastocyst. We propose “prematuration somatic cell fusion” as an approach to generate embryos using somatic cells instead of spermatozoa. The current approach, if improved, would assist production of embryos for couples where the male partner is sterile, however, genetic and chromosomal analysis of the resultant embryos are required before transfer to the mothers.     

Nitrogen and phosphorus addition differentially enhance seed production of dominant species in a temperate steppe

Previous studies have demonstrated changes in plant growth and reproduction in response to nutrient availability, but responses of plant growth and reproduction to multiple levels of nutrient enrichment remain unclear. In this study, a factorial field experiment was performed with manipulation of nitrogen (N) and phosphorus (P) availability to examine seed production of the dominant species, Stipa krylovii, in response to N and P addition in a temperate steppe. There were three levels of N and P addition in this experiment, including no N addition (0 g N m⁻² year⁻¹), low N addition (10 g N m⁻² year⁻¹), and high N addition (40 g N m⁻² year⁻¹) for N addition treatment, and no P addition (0 g P m⁻² year⁻¹), low P addition (5 g P m⁻² year⁻¹), and high P addition (10 g P m⁻² year⁻¹) for P addition treatment. Low N addition enhanced seed production by 814%, 1371%, and 1321% under ambient, low, and high P addition levels, respectively. High N addition increased seed production by 2136%, 3560%, and 3550% under ambient, low, and high P addition levels, respectively. However, P addition did not affect seed production in the absence of N addition, but enhanced it under N addition. N addition enhanced seed production mainly by increasing the tiller number and inflorescence abundance per plant, whereas P addition stimulated it by decreasing the plant density yet stimulating height of plants and their seed number per inflorescence. Our results indicate seed production is not limited by P availability but rather by N availability in the temperate steppe, whereas seed production will be increased by P addition when N availability is improved. These findings enable a better understanding of plant reproduction dynamics in the temperate steppe under intensified nutrient enrichment and can inform their improved management in the future.     

Changes in Glial Cell Line-derived Neurotrophic Factor Expression in the Rostral and Caudal Stumps of the Transected Adult Rat Spinal Cord

Limited information is available regarding the role of endogenous Glial cell line-derived neurotrophic factor (GDNF) in the spinal cord following transection injury. The present study investigated the possible role of GDNF in injured spinal cords following transection injury (T₉–T₁₀) in adult rats. The locomotor function recovery of animals by the BBB (Basso, Beattie, Bresnahan) scale score showed that hindlimb support and stepping function increased gradually from 7 days post operation (dpo) to 21 dpo. However, the locomotion function in the hindlimbs decreased effectively in GDNF-antibody treated rats. GDNF immunoreactivty in neurons in the ventral horn of the rostral stump was stained strongly at 3 and 7 dpo, and in the caudal stump at 14 dpo, while immunostaining in astrocytes was also seen at all time-points after transection injury. Western blot showed that the level of GDNF protein underwent a rapid decrease at 7 dpo in both stumps, and was followed by a partial recovery at a later time-point, when compared with the sham-operated group. GDNF mRNA-positive signals were detected in neurons of the ventral horn, especially in lamina IX. No regenerative fibers from corticospinal tract can be seen in the caudal segment near the injury site using BDA tracing technique. No somatosensory evoked potentials (SEP) could be recorded throughout the experimental period as well. These findings suggested that intrinsic GDNF in the spinal cord could play an essential role in neuroplasticity. The mechanism may be that GDNF is involved in the regulation of local circuitry in transected spinal cords of adult rats.