Dynamic DNA N 6-adenine methylation (6mA) governs the encystment process, showcased in the unicellular eukaryote Pseudocohnilembus persalinus.

The formation of resting cysts commonly found in unicellular eukaryotes is a complex and highly regulated survival strategy against environmental stress that involves drastic physiological and biochemical changes. Although most studies have focused on the morphology and structure of cysts, little is known about the molecular mechanisms that control this process. Recent studies indicate that DNA N 6-adenine methylation (6mA) could be dynamically changing in response to external stimuli; however, its potential role in the regulation of cyst formation remains unknown. We used the ciliate Pseudocohnilembus persalinus, which can be easily induced to form cysts to investigate the dynamic pattern of 6mA in trophonts and cysts. Single-molecule real-time (SMRT) sequencing reveals high levels of 6mA in trophonts that decrease in cysts, along with a conversion of symmetric 6mA to asymmetric 6mA. Further analysis shows that 6mA, a mark of active transcription, is involved in altering the expression of encystment-related genes through changes in 6mA levels and 6mA symmetric-to-asymmetric conversion. Most importantly, we show that reducing 6mA levels by knocking down the DNA 6mA methyltransferase PpAMT1 accelerates cyst formation. Taken together, we characterize the genome-wide 6mA landscape in P. persalinus and provide insights into the role of 6mA in gene regulation under environmental stress in eukaryotes. We propose that 6mA acts as a mark of active transcription to regulate the encystment process along with symmetric-to-asymmetric conversion, providing important information for understanding the molecular response to environmental cues from the perspective of 6mA modification.

Research on the application of Thelephora ganbajun exopolysaccharides in antioxidant, anti-inflammatory and spot-fading cosmetics.

Thelephora ganbajun exopolysaccharides (TGEP) with a "coral-like" branched chain structure (main chain diameter âˆ¼ 80 nm) were prepared by liquid fermentation and fractionated by ion-exchange chromatography. The main fraction (TGEP-2) with the highest in vitro antioxidant capacity was composed of Glc, Man, Gal, GalA, GlcA, Ara, Rha, GlcN, Fuc and Rib in a molar ratio of 465.43:420.43:219.14:188.43:37:35.14:31.43:19.43:11.14:1, with a molecular weight of 1.879 × 104 Da. The sequence of monosaccharide residue release revealed that Gal, Glc and Ara residues were more distributed in the side-branch chains and at their ends, whereas Man and GalA residues were more distributed in the main chains. TGEP-2 contained linear residues (mainly →4)-Glcp-(1 â†’ and →4)-Manp-(1→), branch residues (→3,6)-Glcp-(1→, →4,6)-Glcp-(1 â†’ and →3,6)-Galp-(1→) and terminal residues (Galp-(1→, Manp-(1 â†’ and Glcp-(1→). TGEP-2 consisted of α- and ß-glycosidically linked pyranosides, with a triple helical conformation and many long branches. Zebrafish oxidative stress and inflammation models found that TGEP-2 had antioxidant and anti-inflammatory activities. The zebrafish skin black spot assay showed that TGEP-2 inhibited melanin formation. Therefore, extracellular polysaccharides of T. ganbajun have strong application potential in anti-oxidant, anti-inflammatory and skin spot-fading functions cosmetics.

Effects of Liuzijue Qigong Posture on Aerodynamics of Phonation in Healthy Volunteers.

OBJECTIVES: To verify the possible function of Liuzijue Qigong (LQG), a kind of traditional Chinese health exercise, in improving phonation. METHODS: A total of 30 healthy volunteers (10 males, 20 females) without voice disorders were included. The subjects were asked to have phonation tasks at the sitting and LQG postures. Aerodynamic, electroglottographic, and acoustic parameters were measured. Expiratory Volume (FVC); Subglottic Pressure at comfortable phonation (SGP), Glottal Resistance (GR), Glottal Efficiency (GE); Contact Quotient (CQ), Mean Flow (MF), Fundamental frequency (F0), Mean Sound Pressure Level (SPL); Phonation Threshold Pressure (PTP); and Maximum Phonation Time (MPT) were measured and analyzed. RESULTS: In total subjects, the analysis showed a significant increase in FVC (P = 0.020), SGP (P = 0.043), F0 (P = 0.021), and PTP (P = 0.000) at the LQG posture when compared with the sitting posture, and there is no difference in CQ, MF, SPL, GR, GE, and MPT. CONCLUSIONS: The results showed LQG posture increased the respiratory support and glottal closure, while induced the respiratory system and vocal system in coordination to improve phonation. It is logical to postulate that LQG has potential in the management of voice disorders with glottal closure insufficiency.

New contribution to epigenetic studies: Isolation of micronuclei with high purity and DNA integrity in the model ciliated protist, Tetrahymena thermophila.

The ciliated protist Tetrahymena thermophila is a well-known model organism with typical nuclear dimorphism containing a somatic macronucleus (MAC) and a germline micronucleus (MIC). The presence in the same cell compartment of two nuclei with distinctly different structural and functional properties provides an ideal model system to explore mechanisms of genome maintenance. Although methods for the isolation of MIC have been available for many years, cross-contamination and DNA degradation remain unresolved. Here, we describe a reliable and quick method to isolate MIC with high purity and DNA integrity in T. thermophila. Different factors are examined to optimize the MIC purification. The MAC contamination ratio in purified MIC is about 0.19% and DNA integrity of purified MIC is maintained. We also establish a more accurate method to detect the contamination rate of nuclei including microscopic observation and PCR detection. This study will facilitate further epigenetic research in Tetrahymena.

An Optimized and Versatile Counter-Flow Centrifugal Elutriation Workflow to Obtain Synchronized Eukaryotic Cells.

Cell synchronization is a powerful tool to understand cell cycle events and its regulatory mechanisms. Counter-flow centrifugal elutriation (CCE) is a more generally desirable method to synchronize cells because it does not significantly alter cell behavior and/or cell cycle progression, however, adjusting specific parameters in a cell type/equipment-dependent manner can be challenging. In this paper, we used the unicellular eukaryotic model organism, Tetrahymena thermophila as a testing system for optimizing CCE workflow. Firstly, flow cytometry conditions were identified that reduced nuclei adhesion and improved the assessment of cell cycle stage. We then systematically examined how to achieve the optimal conditions for three critical factors affecting the outcome of CCE, including loading flow rate, collection flow rate and collection volume. Using our optimized workflow, we obtained a large population of highly synchronous G1-phase Tetrahymena as measured by 5-ethynyl-2'-deoxyuridine (EdU) incorporation into nascent DNA strands, bulk DNA content changes by flow cytometry, and cell cycle progression by light microscopy. This detailed protocol can be easily adapted to synchronize other eukaryotic cells.