PbLAC4-like, activated by PbMYB26, related to the degradation of anthocyanin during color fading in pear.

BACKGROUND: Decrease in anthocyanin content results in the loss of red color in leaves, petals and receptacles during development. The content of anthocyanin was affected by the biosynthesis and degradation of anthocyanin. Compared with the known detailed mechanism of anthocyanin biosynthesis, the degradation mechanism is not fully investigated. It is vital to study the degradation mechanism of anthocyanin in pear for promoting the accumulation of anthocyanin and inhibiting the red fading in pear. RESULTS: Here, we reported that laccase encoded by PbLAC4-like was associated with anthocyanin degradation in pear. The expression pattern of PbLAC4-like was negatively correlated with the content of anthocyanin during the color fading process of pear leaves, petals and receptacles. Phylogenetic analysis and sequence alignment revealed that PbLAC4-like played a vital role in anthocyanin degradation. Thus, the degradation of anthocyanin induced by PbLAC4-like was further verified by transient assays and prokaryotic expression. More than 80% of anthocyanin compounds were degraded by transiently over-expressed PbLAC4-like in pear fruitlet peel. The activity of crude enzyme to degrade anthocyanin in leaves at different stages was basically consistent with the expression of PbLAC4-like. The anthocyanin degradation ability of prokaryotic induced PbLAC4-like protein was also verified by enzyme activity assay. Besides, we also identified PbMYB26 as a positive regulator of PbLAC4-like. Yeast one-hybrid and dual luciferase assay results showed that PbMYB26 activated PbLAC4-like expression by directly binding to the PbLAC4-like promoter. CONCLUSIONS: Taken together, the PbLAC4-like activated by PbMYB26, was involved in the degradation of anthocyanin, resulting in the redness fading in different pear tissues.

Isoflurane activates AMP-activated protein kinase to inhibit proliferation, and promote apoptosis and autophagy in cervical carcinoma both in vitro and in vivo.

OBJECTIVE: Isoflurane is an extensively used inhalational anesthesia, and its carcinogenic or anti-cancerous effect has been identified recently. However, the specific role of isoflurane in cervical cancer remains unclear. AIM: This study aimed to investigate the function of isoflurane in cervical cancer as well as the underlying mechanism. METHODS: After isoflurane treatment, HeLa cell viability, percentage of apoptotic cells, expression of active caspase-3/9 were examined by CCK-8 assay, Annexin V-FITC/PI double staining, and Western blot analysis, respectively. ROS generation, ratio of NAD+/NADH, and ATP level after isoflurane stimulation were determined using commercial assay kits. Afterwards, activation of AMPK and autophagy was assessed through Western blot analysis and immunofluorescence. Whether AMPK mediated the isoflurane-induced apoptosis and autophagy was explored by adding an AMPK inhibitor (Compound C). The in vivo function of isoflurane was finally investigated on a HeLa cell xenograft model. RESULTS: Isoflurane inhibited cell viability and induced apoptosis evidenced by upregulation of active caspase-3/9 in HeLa cells. Oxidative stress was triggered by isoflurane, as isoflurane elevated ROS level, and lowered ratio of NAD+/NADH and ATP level. Further results showed isoflurane activated the AMPK/mTOR pathway and induced autophagy. In addition, inhibition of AMPK led to ameliorated effects of isoflurane on apoptosis and autophagy. In vivo experiments proved isoflurane could repress tumorigenesis, activate AMPK, and induce autophagy in Xenograft mouse. CONCLUSIONS: Isoflurane activated AMPK to inhibit proliferation and promote apoptosis and autophagy both in vitro and in vivo.

Analysis of Influencing Factors of Serum Stress Index and Prognosis of HICH Patients by Different Anesthesia Methods Combined with Small Bone Window Microsurgery.

In order to investigate the effects of sevoflurane on the serum stress index level and prognosis of patients with hypertensive cerebral hemorrhage (HICH) during small bone window microsurgery, a total of 102 HICH patients are selected for analysis. MAP values in both groups decreased significantly at T1 and T2 (P < 0.05), and the changes in MAP and HR indexes in the sevoflurane combined group were more stable than those in the control group. The time of postoperative awakening in the sevoflurane combined group decreases significantly than the control group (P < 0.001). The levels of T-AOC and GSH-Px in both groups increase significantly after operation, and those in the sevoflurane combined group increase significantly than the control group (P < 0.001). The levels of MDA and 8-OHDG in the sevoflurane combined group decrease significantly than the control group after operation (P < 0.05). Spearman correlation coefficient analysis shows that the levels of T-AOC and GSH-Px are negatively correlated with the prognosis of HICH patients, while MDA and 8-OHDG are positively correlated with the prognosis of HICH patients (P < 0.001). Sevoflurane interventional anesthesia has a high anesthetic effect in small bone window microsurgery, which has positive effects on controlling blood pressure of HICH patients, shortening postoperative recovery time and improving patients' stress response and neurological function. This paper conducts an in-depth analysis of the prognosis of HICH patients, indicating that the prognosis of HICH patients is closely related to their serum stress indicators T-AOC, GSH-Px, MDA, and 8-OHDG, providing a new direction for follow-up clinical diagnosis and treatment of HICH patients and accurate prognosis assessment.

Effects of Dexmedetomidine Combined with Intravenous Anesthesia on Oxidative Stress Index, Postoperative Sleep Quality, and Brain Function in HICH Patients.

To investigate the effects of dexmedetomidine combined with intravenous anesthesia on oxidative emergency indicators, postoperative sleep quality, and brain function in patients with hypertensive cerebral hemorrhage (HICH), a total of 285 HICH patients admitted to our hospital from February 2020 to February 2021 were selected. The combined anesthesia group (n = 142) and the control group (n = 143) were established by the random number table method. The control group received conventional intravenous anesthesia, and the combined anesthesia group received dexmedetomidine combined intravenous anesthesia. Two groups of patients before and after operation was observed vital signs, oxidative stress index difference, comparing each time, the change of the two groups of brain function index, adverse reactions occurred between observation group, and the postoperative period of Pittsburgh Sleep Quality Index Scale (PSQI) score as a result, the Pearson correlation coefficient analysis of oxidative stress level and the correlation of HICH patients sleep quality. After operation, the mean arterial pressure (MAP) and heart rate (HR) of patients in both groups decreased significantly. The MAP level in the combined anesthesia group significantly increased compared to the control group, and the HR level decreased significantly than the control group (all P < 0.05). The levels of TNF-α, IL-6 and MDA in both groups increased significantly on day 7 after operation compared with before operation, but the indexes in the combined anesthesia group significantly decreased compared with the control group (P < 0.05). The level of superoxide dismutase (SOD) in both groups significantly decreased compared to that before operation, and the index value in combined anesthesia group significantly increased compared to that in the control group (P < 0.05). After surgery, the levels of central nerve specific protein (S100-ß) and neuron specific enolase (NSE) in 2 groups increased with time, and the indexes in the combined anesthesia group significantly decreased compared to the control group (all P < 0.05). The incidence of adr in combined anesthesia group decreased significantly than that in control group (P < 0.05). After surgery, PSQI scores of the two groups showed a downward trend with time extension, and scores of the combined anesthesia group decreased significantly than those of the control group at 24 h, 48 h and 7 d after surgery (all P < 0.05). Pearson's correlation coefficient was used to analyze that TNF-α, IL-6, and MDA levels were positively correlated with PSQI score, while SOD level was negatively correlated with PSQI score (all P < 0.05). Dexmedetomidine combined with intravenous anesthesia can significantly improve the vital signs and oxidative stress response of HICH patients, effectively reduce the risk of adverse reactions, have little impact on the brain function of patients, and can improve the postoperative sleep quality of patients. This operation is worthy of clinical application. In addition, this study further analyzed the influence mechanism of postoperative sleep quality in patients with HICH and showed that TNF-α, IL-6, MDA, and SOD were all correlated with sleep quality in patients with HICH, suggesting that follow-up detection of these indicators has positive significance in improving the prognosis of patients.

CPPU may induce gibberellin-independent parthenocarpy associated with PbRR9 in 'Dangshansu' pear.

Parthenocarpy is a valuable trait in self-incompatible plants, such as pear. N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU), a synthetic cytokinin analog, can induce parthenocarpy in pear (Pyrus spp.), but the mechanism of induction is unclear. To investigate the role of gibberellin in CPPU-induced parthenocarpy in pear, CPPU supplemented with paclobutrazol (PAC) was sprayed onto 'Dangshansu' pear. We found that the fruit set rate of pear treated with CPPU supplemented with PAC was identical to that in a CPPU-alone treatment group. In regard to cell development, CPPU mainly promoted hypanthium cell division and expansion, and PAC application had no influence on CPPU-induced cell development. RNA sequencing revealed that gibberellin 20 oxidase and gibberellin 3 oxidase genes were not differentially expressed following CPPU treatment. According to the analysis of fruit phytohormone content, the CPPU treatments did not induce gibberellin biosynthesis. These results suggest that CPPU-induced parthenocarpy may be gibberellin independent in 'Dangshansu' pear. After CPPU treatment, the indole acetic acid (IAA) content in fruit was significantly increased, and the abscisic acid (ABA) content was significantly decreased. Similarly, RNA sequencing revealed that many genes involved in the auxin and ABA pathways were significantly differentially expressed in the CPPU treatment groups; among them, indole-3-pyruvate monooxygenase (YUCCA) was significantly upregulated and 9-cis-epoxycarotenoid dioxygenase (NCED) was significantly downregulated. IAA and ABA may thus play important roles in CPPU-induced parthenocarpy. PbTwo-component response regulator9 (PbRR9), PbYUCCA4, and PbNCED6 were then selected to further elucidate the mechanism of CPPU-induced parthenocarpy. A yeast one-hybrid assay indicated that PbRR9 can combine with the PbYUCCA4 and PbNCED6 promoters. Dual luciferase assays revealed that PbRR9 can promote and repress the activities of the PbYUCCA4 and PbNCED6 promoters, respectively. After the transient expression of PbRR9 in fruits, PbYUCCA4 expression was significantly upregulated, and PbNCED6 expression was significantly downregulated. This study uncovered a CPPU-induced parthenocarpy mechanism that is different from that in tomato. CPPU may upregulate PbYUCCA4 and downregulate PbNCED6 by upregulating PbRR9, thereby increasing IAA content and decreasing ABA content to ultimately induce parthenocarpy in 'Dangshansu' pear. However, because only a single time point was used and because 'botanical' and 'accessory' fruits have different structures, this conclusion is still preliminary.

Jasmonate and Ethylene-Regulated Ethylene Response Factor 22 Promotes Lanolin-Induced Anthocyanin Biosynthesis in 'Zaosu' Pear (Pyrusbretschneideri Rehd.) Fruit.

Anthocyanin contributes to the coloration of pear fruit and enhances plant defenses. Members of the ethylene response factor (ERF) family play vital roles in hormone and stress signaling and are involved in anthocyanin biosynthesis. Here, PbERF22 was identified from the lanolin-induced red fruit of 'Zaosu' pear (Pyrus bretschneideri Rehd.) using a comparative transcriptome analysis. Its expression level was up- and down-regulated by methyl jasmonate and 1-methylcyclopropene plus lanolin treatments, respectively, which indicated that PbERF22 responded to the jasmonate- and ethylene-signaling pathways. In addition, transiently overexpressed PbERF22 induced anthocyanin biosynthesis in 'Zaosu' fruit, and a quantitative PCR analysis further confirmed that PbERF22 facilitated the expression of anthocyanin biosynthetic structural and regulatory genes. Moreover, a dual luciferase assay showed that PbERF22 enhanced the activation effects of PbMYB10 and PbMYB10b on the PbUFGT promoter. Therefore, PbERF22 responses to jasmonate and ethylene signals and regulates anthocyanin biosynthesis. This provides a new perspective on the correlation between jasmonate-ethylene crosstalk and anthocyanin biosynthesis.

Evaluation of three tracers for labeling distal cerebrospinal fluid-contacting neurons.

It has been reported that distal cerebrospinal fluid-contacting neurons (dCSF-CNs) can be detected by immunohistochemical assay using cholera toxin subunit B-conjugated horseradish peroxidase (CB-HRP). In the present study, another two methods with CB alone or CB-conjugated FITC (CB-FITC) were used, and the results from the three methods were compared. Adult Sprague-Dawley rats were randomly divided into three groups with CB-HRP, CB or CB-FITC. Tracers were diluted to 30% in artificial cerebrospinal fluid and injected separately (in a volume of 3 µL) into the lateral ventricle. Animals from the CB-HRP and CB groups were perfused 48 h after surgery while animals from the CB-FITC group were perfused 1, 3, 6, 12, 24 or 48 h after surgery. The brain was sectioned (40 µm) for immunofluorescence and five sections with positive neurons were selected from each rat for neuron counting. Three clusters of positive neurons in a 'Y-like' distribution were detected ventral to the cerebral aqueduct of rats from the three groups. No significant difference was observed among the quantitative data. In the CB-FITC group, stable staining was detected even at 6 h after injection. Taken together, lateral ventricle injection of CB/CB-FITC is a useful method for labeling dCSF-CNs in rats. The CB-FITC method makes dCSF-CNs labeling much simpler and more convenient.