Exploring the RNA Editing Events and Their Potential Regulatory Roles in Tea Plant (Camellia sinensis L.).

RNA editing is a post-transcriptional modification process that alters the RNA sequence relative to the genomic blueprint. In plant organelles (namely, mitochondria and chloroplasts), the most common type is C-to-U, and the absence of C-to-U RNA editing results in abnormal plant development, such as etiolation and albino leaves, aborted embryonic development and retarded seedling growth. Here, through PREP, RES-Scanner, PCR and RT-PCR analyses, 38 and 139 RNA editing sites were identified from the chloroplast and mitochondrial genomes of Camellia sinensis, respectively. Analysis of the base preference around the RNA editing sites showed that in the -1 position of the edited C had more frequent occurrences of T whereas rare occurrences of G. Three conserved motifs were identified at 25 bases upstream of the RNA editing site. Structural analyses indicated that the RNA secondary structure of 32 genes, protein secondary structure of 37 genes and the three-dimensional structure of 5 proteins were altered due to RNA editing. The editing level analysis of matK and ndhD in six tea cultivars indicated that matK-701 might be involved in the color change of tea leaves. Furthermore, 218 PLS-CsPPR proteins were predicted to interact with the identified RNA editing sites. In conclusion, this study provides comprehensive insight into RNA editing events, which will facilitate further study of the RNA editing phenomenon of the tea plant.

Diagnosis and treatment of novel coronavirus pneumonia based on the theory of traditional Chinese medicine / 中西医结合学报

Since the outbreak of novel coronavirus pneumonia (coronavirus disease 2019, COVID-19), it has rapidly spread to 187 countries, causing serious harm to the health of people and a huge social burden. However, currently, drugs specifically approved for clinical use are not available, except for vaccines against COVID-19 that are being evaluated. Traditional Chinese medicine (TCM) is capable of performing syndrome differentiation and treatment according to the clinical manifestations of patients, and has a better ability of epidemic prevention and control. The authors comprehensively analyzed the etiology and pathogenesis of COVID-19 based on the theory of TCM, and discussed its syndrome differentiation, treatment and prevention measures so as to provide strategies and reference for the prevention and treatment with TCM.

[Engineering of a flavonoid 3'-hydroxylase from tea plant (Camellia sinensis) for biosynthesis of B-3',4'-dihydroxylated flavones].

Objective: A flavonoid 3'-hydroxylase from tea plant was engineered to synthesize B-3',4'-dihydroxylated flavones such as eriodictyol, dihydroquercetin and quercetin. Methods: Four articifical P450 constructs harboring both flavonoid 3'-hydroxylase gene from Camellia sinensis (CsF3'H) and P450 reductase gene from Arabidopsis thaliana (ATR1 or ATR2) were introduced into Escherichia coli strains TOP10, DH5α and BL21, resultantly engineering strains S1 to S12. The plasmid pYES-Dest52-CsF3'H harboring CsF3'H gene was introduced into yeast Saccharomyces cerevisiae WAT11 designated as strain S13. The plasmid pES-HIS-CsF3H::AtFLS 9 AA was constructed through fusing flavanone 3-hydroxylase gene from Camellia sinensis (CsF3H) and flavonol synthase gene from Arabidopsis thaliana (AtFLS). Plasmid pES-URA-CsF3'H and pES-HIS-CsF3H::AtFLS 9 AA were then co-introduced into yeast S. cerevisiae WAT11 designated as strain S14. Results: Strain S6 generated highest bioconversion efficiency at 25℃ among all E. coli strains during 24 h fernentation. Supplemented with 1000 µmol/L naringenin, dihydrokaempferol and kaempferol, the maximum amounts of eriodictyol, dihydroquercetin and quercetin produced by strain S13 were 734.32 µmol/L, 446.07 µmol/L and 594.64 µmol/L respectively. Supplemented with 5 mmol/L naringenin, the maximum amounts of eriodictyol, kaempferol, quercetin, dihydroquercetin and dihydrokaempferol produced by strain S14 were 1412.16 µmol/L, 490.25 µmol/L, 445.75 µmol/L, 66.75 µmol/L and 73.50 µmol/L during 36-48 h fermentaion respectively. Conclusion: CsF3'H was engineered for biosynthesis of B-3',4'-dihydroxylated flavone.

Melatonin promotes development of haploid germ cells from early developing spermatogenic cells of Suffolk sheep under in vitro condition.

Promotion of spermatogonial stem cell (SSC) differentiation into functional sperms under in vitro conditions is a great challenge for reproductive physiologists. In this study, we observed that melatonin (10(-7) M) supplementation significantly enhanced the cultured SSCs differentiation into haploid germ cells. This was confirmed by the expression of sperm special protein, acrosin. The rate of SSCs differentiation into sperm with melatonin supplementation was 11.85 ± 0.93% which was twofold higher than that in the control. The level of testosterone, the transcriptions of luteinizing hormone receptor (LHR), and the steroidogenic acute regulatory protein (StAR) were upregulated with melatonin treatment. At the early stage of SSCs culture, melatonin suppressed the level of cAMP, while at the later stage, it promoted cAMP production. The similar pattern was observed in testosterone content. Expressions for marker genes of meiosis anaphase, Dnmt3a, and Bcl-2 were upregulated by melatonin. In contrast, Bax expression was downregulated. Importantly, the in vitro-generated sperms were functional and they were capable to fertilize oocytes. These fertilized oocytes have successfully developed to the blastula stage.

Huperzine A activates Wnt/ß-catenin signaling and enhances the nonamyloidogenic pathway in an Alzheimer transgenic mouse model.

Huperzine A (HupA) is a reversible and selective inhibitor of acetylcholinesterase (AChE), and it has multiple targets when used for Alzheimer's disease (AD) therapy. In this study, we searched for new mechanisms by which HupA could activate Wnt signaling and reduce amyloidosis in AD brain. A nasal gel containing HupA was prepared. No obvious toxicity of intranasal administration of HupA was found in mice. HupA was administered intranasally to ß-amyloid (Aß) precursor protein and presenilin-1 double-transgenic mice for 4 months. We observed an increase in ADAM10 and a decrease in BACE1 and APP695 protein levels and, subsequently, a reduction in Aß levels and Aß burden were present in HupA-treated mouse brain, suggesting that HupA enhances the nonamyloidogenic APP cleavage pathway. Importantly, our results further showed that HupA inhibited GSK3α/ß activity, and enhanced the ß-catenin level in the transgenic mouse brain and in SH-SY5Y cells overexpressing Swedish mutation APP, suggesting that the neuroprotective effect of HupA is not related simply to its AChE inhibition and antioxidation, but also involves other mechanisms, including targeting of the Wnt/ß-catenin signaling pathway in AD brain.

Zinc overload enhances APP cleavage and Aß deposition in the Alzheimer mouse brain.

BACKGROUND: Abnormal zinc homeostasis is involved in ß-amyloid (Aß) plaque formation and, therefore, the zinc load is a contributing factor in Alzheimer's disease (AD). However, the involvement of zinc in amyloid precursor protein (APP) processing and Aß deposition has not been well established in AD animal models in vivo. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, APP and presenilin 1 (PS1) double transgenic mice were treated with a high dose of zinc (20 mg/ml ZnSO4 in drinking water). This zinc treatment increased APP expression, enhanced amyloidogenic APP cleavage and Aß deposition, and impaired spatial learning and memory in the transgenic mice. We further examined the effects of zinc overload on APP processing in SHSY-5Y cells overexpressing human APPsw. The zinc enhancement of APP expression and cleavage was further confirmed in vitro. CONCLUSIONS/SIGNIFICANCE: The present data indicate that excess zinc exposure could be a risk factor for AD pathological processes, and alteration of zinc homeostasis is a potential strategy for the prevention and treatment of AD.

Oral administration of Crataegus flavonoids protects against ischemia/reperfusion brain damage in gerbils.

Stroke is the third leading cause of death as dementia is a main symptom of Alzheimer's disease. One of the important mechanisms in the pathogeny of stroke is free radical production during the reperfusion period, therefore the effects of a type of natural antioxidant, i.e. Crataegus flavonoids (CF), on brain ischemic insults were investigated in Mongolian gerbil stroke model. Results showed that pretreatment of the animals with CF decreased reactive oxygen species (ROS) production, thiobarbituric acid reactive substances content, and nitrite/nitrate concentration in brain homogenate, increased the brain homogenate-associated antioxidant level in a dose-dependent manner. CF pretreatment increased the amount of biologically available NO by scavenging of superoxide anion produced during reperfusion. At same time, in the process of ischemia/reperfusion brain damage, the content of nitrite/nitrate (the end product of NO) increased, and of NO detected by ESR decreased. Oral pretreatment with CF decreased the nitrite/nitrate content in the brain homogenate and increased the biologically available NO concentration in a dose-dependent manner. The increasing effect of antioxidant on NO might be due to its scavenging effect on superoxide anion, which could react with NO into peroxynitrite. iNOS was implied in delayed neuron death after brain ischemic damage and it was found that pretreatment with CF could decrease the protein level of tumor necrosis factor (TNF)-alpha and nuclear factor-kappa B (NF-kappaB), and increase the mRNA level of NOS estimated by western blotting and RT-PCR. More neurons survived and fewer cells suffered apoptosis in the hippocampal CA1 region of CF treated animal brain. These results suggest that oral administration of this antioxidant increases the antioxidant level in the brain and protects the brain against delayed cell death caused by ischemia/reperfusion injury.

Protective effects of green tea polyphenols on human HepG2 cells against oxidative damage of fenofibrate.

The aim of this work was to investigate the protective effects of green tea polyphenols on the cytotoxic effects of hypolipidemic agent fenofibrate (FF), a peroxisome proliferator (PP), in human HepG2 cells. The results showed that high concentrations of FF induced human HepG2 cell death through a mechanism involving an increase of reactive oxygen species (ROS) and intracellular reduced glutathione (GSH) depletion. These effects were partially prevented by antioxidant green tea polyphenols. The elevated expression of PP-activated receptors alpha (PPARalpha) in HepG2 cells induced by FF was also decreased by treatment with green tea polyphenols. In conclusion, this result demonstrates that oxidative stress and PPARalpha are involved in FF cytotoxicity and green tea polyphenols have a protective effect against FF-induced cellular injury. It may be beneficial for the hyperlipidemic patients who were administered the hypolipidemic drug fenofibrate to drink tea or use green tea polyphenols synchronously during their treatment.