Embryonic diapause due to high glucose is related to changes in glycolysis and oxidative phosphorylation, as well as abnormalities in the TCA cycle and amino acid metabolism

IntroductionThe adverse effects of high glucose on embryos can be traced to the preimplantation stage. This study aimed to observe the effect of high glucose on early-stage embryos. Methods and resultsSeven-week-old ICR female mice were superovulated and mated, and the zygotes were collected. The zygotes were randomly cultured in 5 different glucose concentrations (control, 20mM, 40mM, 60mM and 80mM glucose). The cleavage rate, blastocyst rate and total cell number of blastocyst were used to assess the embryo quality. 40 mM glucose was selected to model high glucose levels in this study. 40mM glucose arrested early embryonic development, and the blastocyst rate and total cell number of the blastocyst decreased significantly as glucose concentration was increased. The reduction in the total cell number of blastocysts in the high glucose group was attributed to decreased proliferation and increased cell apoptosis, which is associated with the diminished expression of GLUTs (GLUT1, GLUT2, GLUT3). Furthermore, the metabolic characterization of blastocyst culture was observed in the high-glucose environment. DiscussionThe balance of glycolysis and oxidative phosphorylation at the blastocyst stage was disrupted. And embryo development arrest due to high glucose is associated with changes in glycolysis and oxidative phosphorylation, as well as abnormalities in the TCA cycle and amino acid metabolism

Comparative pilot study on the effects of pulsating and static cupping on non-specific neck pain and local skin blood perfusion

Objective: To compare the effects of pulsating and static cupping on non-specific neck pain and local skin microcirculation blood perfusion, which is a pilot study. Methods: Seventy participants with non-specific neck pain were randomized to the following groups: low-frequency pulsating cupping (LF, n = 20); high-frequency pulsating cupping (HF, n = 20); static cupping (SC, n = 20), or waiting list (WL, n = 10). The LF, HF, and SC received a bilateral 10-minute cupping treatment at Jianzhongshu (SI 15). Outcomes were pain intensity (visual analog scale, VAS), functional status (Neck Disability Index, NDI), and skin blood perfusion at the SI 15, Dazhui (GV 14), and Shenzhu (GV 12) acupoint areas, measured using Laser Speckle Contrast Analysis technology. Results: Both LF and HF groups showed a significant reduction in VAS scores compared with the SC group (9.00, 95% CI 1.05–16.95, P = .027; 8.75, 95% CI 0.80–16.70, P = .031). There was no significant difference in VAS scores between the LF and HF groups (P > .05) and between NDI scores measured 3 days after intervention among the four groups (P > .05). In the SI 15 area, blood perfusion in the three treatment groups was higher than that in WL group (P < .01), and the perfusion unit (PU) of the HF pulsating group at 5 minutes after intervention was significantly higher compared with the SC group (P < .05). In the GV 14 area, blood perfusion in the two pulsating cupping groups was higher compared with the WL and SC groups after cupping (P < .05). In the GV 12 area, the PU of the LF group was higher compared with the other three groups only at the time of cup removal (P < .05). Conclusion: This study showed that pulsating cupping may have more favorable analgesic effects on non-specific neck pain compared with static cupping, which may be related to its better effect on improving the local skin blood perfusion. Keywords: Pulsating cupping, Static cupping, Non-specific neck pain, VAS, Blood perfusio

Production of Cannabidiol Acid Synthase by Chassis Pichia pastoris and of Its Catalytic Activity Analysis

In order to obtain the higher expression of cannabinoid acid synthase (CBDAS) through the microbial chassis, the recombinant enzyme for the in vitro synthesis of cannabinoid (CBD) was studied. The CBDAS gene was firstly cloned from cannabis leaves with higher content of CBD, its basic physicochemical properties of proteins were then analyzed by bioinformatics methods. After construction of the recombinant plasmid pPIC9K-CBDAS and transformation into Pichia pastoris, conditions of the recombinant protein CBDAS were optimized and its catalytic activities were finally analyzed. The results indicated that the constructed pPIC9K-CBDAS fusion protein expression system could be successfully expressed in P. pastoris. Under conditions of 1% of methanol addition, pH6.0 of medium, 48 h of induction, the maximum expression was obtained. At 4 h reaction, the product of cannabigerol acid (CBGA) was significantly increased, in which attributed to catalysis of substrate CBGA by the recombinant CBDAS. In the following, contents of CBD were significantly increased at 8 h (P<0.05) and those of CBDA and CBD were reached to the maximum at 12 h. After reactions at 12 h in the crude extract of cannabis leaf and the standardized CBGA, CBDAS could catalyze to product 60.64 and 20.12 ng/mL of CBDA, 128.01 and 207.87 ng/mL of CBD, respectively, in which CBDAS indicated a stronger catalytic activity. In conclusion, heterologous recombinant expression of cannabis CBDAS was achieved through yeast chassis, which provided active enzymes for the efficient synthesis of CBDA and CBD

C9orf72 poly(PR) aggregation in nucleus induces ALS/FTD-related neurodegeneration in cynomolgus monkeys

Poly(PR) is a dipeptide repeat protein comprising proline and arginine residues. It is one of the translational product of expanded G4C2 repeats in the C9orf72 gene, and its accumulation is contributing to the neuropathogenesis of C9orf72-associated amyotrophic lateral sclerosis and/or frontotemporal dementia (C9-ALS/FTD). In this study, we demonstrate that poly(PR) protein alone is sufficient to induce neurodegeneration related to ALS/FTD in cynomolgus monkeys. By delivering poly(PR) via AAV, we observed that the PR proteins were located within the nucleus of infected cells. The expression of (PR)50 protein, consisting of 50 PR repeats, led to increased loss of cortical neurons, cytoplasmic lipofuscin, and gliosis in the brain, as well as demyelination and loss of ChAT positive neurons in the spinal cord of monkeys. While, these pathologies were not observed in monkeys expressing (PR)5, a protein comprising only 5 PR repeats. Furthermore, the (PR)50-expressing monkeys exhibited progressive motor deficits, cognitive impairment, muscle atrophy, and abnormal electromyography (EMG) potentials, which closely resemble clinical symptoms seen in C9-ALS/FTD patients. By longitudinally tracking these monkeys, we found that changes in cystatin C and chitinase-1 (CHIT1) levels in the cerebrospinal fluid (CSF) corresponded to the phenotypic progression of (PR)50-induced disease. Proteomic analysis revealed that the major clusters of dysregulated proteins were nuclear-localized, and downregulation of the MECP2 protein was implicated in the toxic process of poly(PR). This research indicates that poly(PR) expression alone induces neurodegeneration and core phenotypes associated with C9-ALS/FTD in monkeys, which may provide insights into the mechanisms of disease pathogenesis