Facile fabrication of Ti4+-immobilized magnetic nanoparticles by phase-transitioned lysozyme nanofilms for enrichment of phosphopeptides.

In this study, titanium (IV)-immobilized magnetic nanoparticles (Ti4+-PTL-MNPs) were firstly synthesized via a one-step aqueous self-assembly of lysozyme nanofilms for efficient phosphopeptide enrichment. Under physiological conditions, lysozymes readily self-organized into phase-transitioned lysozyme (PTL) nanofilms on Fe3O4@SiO2 and Fe3O4@C MNP surfaces with abundant functional groups, including -NH2, -COOH, -OH, and -SH, which can be used as multiple linkers to efficiently chelate Ti4+. The obtained Ti4+-PTL-MNPs possessed high sensitivity of 0.01 fmol µL-1 and remarkable selectivity even at a mass ratio of ß-casein to BSA as low as 1:400 for phosphopeptide enrichment. Furthermore, the synthesized Ti4+-PTL-MNPs can also selectively identify low-abundance phosphopeptides from extremely complicated human serum samples and their rapid separation, good reproducibility, and excellent recovery were also proven. This one-step self-assembly of PTL nanofilms facilitated the facile and efficient surface functionalization of various nanoparticles for proteomes/peptidomes.

Bifunctional MNPs@UIO-66-Arg core-shell-satellite nanocomposites for enrichment of phosphopeptides.

A facile and mild method based on self-assembled lysozyme (LYZ) to fabricate bifunctional MNPs@UIO-66-Arg core-shell-satellite nanocomposites (CSSNCs) is reported for the high-efficiency enrichment of phosphopeptides. Under physiological conditions, LYZ rapidly self-assembled into a robust coating on Fe3O4@SiO2 magnetic nanoparticles (MNPs) with abundant surface functional groups, which effectively mediate heterogeneous nucleation and growth of UIO-66 nanocrystals. Well-defined MNPs@UIO-66 CSSNCs with stacked pores, showing high specific surface area (333.65 m2 g- 1) and low mass transfer resistance, were successfully fabricated by fine-tuning of the reaction conditions including reaction time and acetic acid content. Furthermore, the UIO-66 shells were further modified with arginine to obtain bifunctional MNPs@UIO-66-Arg CSSNCs. Thanks to the unique morphology and synergistic effect of Zr-O clusters and guanidine groups, the bifunctional MNPs@UIO-66-Arg CSSNCs exhibited outstanding enrichment performance for phosphopeptides, delivering a low limit of detection (0.1 fmol), high selectivity (ß-casein/BSA, mass ratio 1:2000), and good capture capacity (120 mg g- 1). The mechanism for phosphopeptides capture may attribute to the hydrogen bonds, electrostatic interactions, and Zr-O-P bonds between phosphate groups in peptides and guanidyl/Zr-O clusters on bifunctional MNPs@UIO-66-Arg CSSNCs. In addition, the small stacking pores on the core-shell-satellite architecture may selectively capture phosphopeptides with low molecular weight, eliminating interference of other large molecular proteins in complex biological samples.

Delirium in older patients given propofol or sevoflurane anaesthesia for major cancer surgery: a multicentre randomised trial.

BACKGROUND: Delirium is a common and disturbing postoperative complication that might be ameliorated by propofol-based anaesthesia. We therefore tested the primary hypothesis that there is less delirium after propofol-based than after sevoflurane-based anaesthesia within 7 days of major cancer surgery. METHODS: This multicentre randomised trial was conducted in 14 tertiary care hospitals in China. Patients aged 65-90 yr undergoing major cancer surgery were randomised to either propofol-based anaesthesia or to sevoflurane-based anaesthesia. The primary endpoint was the incidence of delirium within 7 postoperative days. RESULTS: A total of 1228 subjects were enrolled and randomised, with 1195 subjects included in the modified intention-to-treat analysis (mean age 71 yr; 422 [35%] women); one subject died before delirium assessment. Delirium occurred in 8.4% (50/597) of subjects given propofol-based anaesthesia vs 12.4% (74/597) of subjects given sevoflurane-based anaesthesia (relative risk 0.68 [95% confidence interval {CI}: 0.48-0.95]; P=0.023; adjusted relative risk 0.59 [95% CI: 0.39-0.90]; P=0.014). Delirium reduction mainly occurred on the first day after surgery, with a prevalence of 5.4% (32/597) with propofol anaesthesia vs 10.7% (64/597) with sevoflurane anaesthesia (relative risk 0.50 [95% CI: 0.33-0.75]; P=0.001). Secondary endpoints, including ICU admission, postoperative duration of hospitalisation, major complications within 30 days, cognitive function at 30 days and 3 yr, and safety outcomes, did not differ significantly between groups. CONCLUSIONS: Delirium was a third less common after propofol than sevoflurane anaesthesia in older patients having major cancer surgery. Clinicians might therefore reasonably select propofol-based anaesthesia in patients at high risk of postoperative delirium. CLINICAL TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR-IPR-15006209) and ClinicalTrials.gov (NCT02662257).

Long-term survival in older patients given propofol or sevoflurane anaesthesia for major cancer surgery: follow-up of a multicentre randomised trial.

BACKGROUND: Experimental evidence indicates that i.v. anaesthesia might reduce cancer recurrence compared with volatile anaesthesia, but clinical information is observational only. We therefore tested the primary hypothesis that propofol-based anaesthesia improves survival over 3 or more years after potentially curative major cancer surgery. METHODS: This was a long-term follow-up of a multicentre randomised trial in 14 tertiary hospitals in China. We enrolled 1228 patients aged 65-90 yr who were scheduled for major cancer surgery. They were randomised to either propofol-based i.v. anaesthesia or to sevoflurane-based inhalational anaesthesia. The primary endpoint was overall survival after surgery. Secondary endpoints included recurrence-free and event-free survival. RESULTS: Amongst subjects randomised, 1195 (mean age 72 yr; 773 [65%] male) were included in the modified intention-to-treat analysis. At the end of follow-up (median 43 months), there were 188 deaths amongst 598 patients (31%) assigned to propofol-based anaesthesia compared with 175 deaths amongst 597 patients (29%) assigned to sevoflurane-based anaesthesia; adjusted hazard ratio 1.02; 95% confidence interval (CI): 0.83-1.26; P=0.834. Recurrence-free survival was 223/598 (37%) in patients given propofol anaesthesia vs 206/597 (35%) given sevoflurane anaesthesia; adjusted hazard ratio 1.07; 95% CI: 0.89-1.30; P=0.465. Event-free survival was 294/598 (49%) in patients given propofol anaesthesia vs 274/597 (46%) given sevoflurane anaesthesia; adjusted hazard ratio 1.09; 95% CI 0.93 to 1.29; P=0.298. CONCLUSIONS: Long-term survival after major cancer surgery was similar with i.v. and volatile anaesthesia. Propofol-based iv. anaesthesia should not be used for cancer surgery with the expectation that it will improve overall or cancer-specific survival. CLINICAL TRIAL REGISTRATIONS: ChiCTR-IPR-15006209; NCT02660411.

Stress response and signalling of a low-temperature bioaugmentation system in decentralized wastewater treatment: Degradation characteristics, community structure, and bioaugmented mechanisms.

Low temperatures present challenges for stable wastewater treatment operations in cold regions. Low-temperature effective microorganisms (LTEM) were added as a bioaugmentation strategy at a decentralized treatment facility to improve performance. The effects of a low-temperature bioaugmentation system (LTBS) with LTEM at low temperatures (4 °C) on organic pollutant performance, microbial community changes, and the metabolic pathways of functional genes and functional enzymes were studied. To explore the bioaugmentation mechanism of LTBS based on stress response and signalling. The results showed that the start-up time of the LTBS (S2) with LTEM was shorter (8 days) and that it removed COD and NH4+-N at higher rates (87 % and 72 %, respectively) at 4 °C. LTEM effectively degraded complex macromolecular organics into small molecular organics, and decomposing sludge flocs and the changing the extracellular polymeric substances (EPS) structure removed more organics and nitrogen. LTEM and local microbial communities (nitrifying and denitrifying bacteria) improved the ability of organic matter degradation and denitrification of the LTBS and formed a core microbial community dominated by LTEM (Bacillus and Pseudomonas). Finally, based on the functional enzymes and metabolic pathways of the LTBS, a low-temperature strengthening mechanism consisting of 6 cold stress responses and signal pathways under low temperatures was formed. This study demonstrated that the LTEM-dominated LTBS could provide an engineering alternative for future decentralized wastewater treatment in cold regions.

Transcriptomics and Metabolomics Analyses Reveal Defensive Responses and Flavonoid Biosynthesis of Dracaena cochinchinensis (Lour.) S. C. Chen under Wound Stress in Natural Conditions.

Dracaena cochinchinensis has special defensive reactions against wound stress. Under wound stress, D. cochinchinensis generates a resin that is an important medicine known as dragon's blood. However, the molecular mechanism underlying the defensive reactions is unclear. Metabolomics and transcriptomics analyses were performed on stems of D. cochinchinensis at different timepoints from the short term to the long term after wounding. According to the 378 identified compounds, wound-induced secondary metabolic processes exhibited three-phase characteristics: short term (0-5 days), middle term (10 days-3 months), and long term (6-17 months). The wound-induced transcriptome profile exhibited characteristics of four stages: within 24 h, 1-5 days, 10-30 days, and long term. The metabolic regulation in response to wound stress mainly involved the TCA cycle, glycolysis, starch and sucrose metabolism, phenylalanine biosynthesis, and flavonoid biosynthesis, along with some signal transduction pathways, which were all well connected. Flavonoid biosynthesis and modification were the main reactions against wound stress, mainly comprising 109 flavonoid metabolites and 93 wound-induced genes. A group of 21 genes encoding CHS, CHI, DFR, PPO, OMT, LAR, GST, and MYBs were closely related to loureirin B and loureirin C. Wound-induced responses at the metabolome and transcriptome level exhibited phase characteristics. Complex responses containing primary metabolism and flavonoid biosynthesis are involved in the defense mechanism against wound stress in natural conditions, and flavonoid biosynthesis and modification are the main strategies of D. cochinchinensis in the long-term responses to wound stress.

Bioaugmentation of quinoline-degrading bacteria for coking wastewater treatment: performance and microbial community analysis.

Ochrobactrum sp. XKL1, previously found to have the ability to efficiently degrade quinoline, was bioaugmented into a lab-scale A/O/O system to treat real coking wastewater. During the bioaugmentation stage, the removal of quinoline and pyridine of the O1 tank could be enhanced by 9.88% and 7.96%, respectively. High-throughput sequencing analysis indicated that the addition of XKL1 could significantly affect the alteration of microbial community structure in the sludge. In addition, the relative abundance of Ochrobactrum has demonstrated a trend of increasing first followed by decreasing with the highest abundance of 7.87% attained on the 94th day. The bioaugmentation effects lasted for about 14 days after the strains was inoculated into the reactor. Although a decrease in the relative abundance of XKL1 was observed for a rather short period of time, the bioaugmented A/O/O system has been proven to be more effective in the removal of organic pollutants than the control. Hence, the results of this study indicated that the bioaugmentation with XKL1 is a feasible operational strategy that would be able to enhance the removal of NHCs in the treatment of coking wastewater with complex composition and high organic concentrations.

Vector magnetic field sensor based on U-bent single-mode fiber and magnetic fluid.

A novel, compact, and easy fabrication vector magnetic field sensor has been proposed and investigated. The proposed sensor consists of a U-bent single-mode fiber fixed in a magnetic-fluid-filled vessel. Neither mechanical modification nor additional fiber grating is needed during the sensor fabrication. The results show that the response of magnetic fluid to magnetic field can be used to measure the direction and intensity of magnetic field via whispering gallery modes supported by the U-bent fiber structure with suitable bending radius. The sensitivity of direction is 0.251 nm/°, and the maximum magnetic field intensity sensitivity is 0.517 nm/mT. Besides, the results of this work prove the feasibility for realizing vector magnetic sensors based on other bending structures (such as bending multimode interference, bending SPR structure) in the future.

The response of polycyclic aromatic hydrocarbon degradation in coking wastewater treatment after bioaugmentation with biosurfactant-producing bacteria Pseudomonas aeruginosa S5.

The polycyclic aromatic hydrocarbons (PAHs) that accumulate during the coking wastewater treatment process are hazardous for the surrounding environment. High molecular weight (HMW) PAHs account for more than 85% of the total PAHs in coking wastewater and sludge, respectively. The degradation of total PAHs increased by 18.97% due to the increased bioavailability of PAHs, after the biosurfactant-producing bacteria Pseudomonas aeruginosa S5 was added. The toxicity of total PAHs to humans was reduced by 26.66% after inoculation with S5. The results suggest biosurfactant-producing bacteria Pseudomonas aeruginosa S5 not only increase the biodegradation of PAHs significantly, but also have a better effect on reducing the human toxicity of PAHs. Kinetic analyses show that PAHs biodegradation fits to first-order kinetics. The degradation rate constant (k) value decreases as the number of PAH rings increases, indicating that HMW PAHs are more difficult to be biodegraded than low molecular weight (LMW) PAHs. The results indicate the bioaugmentation with the biosurfactant-producing strain has significant potential and utility in remediation of PAHs-polluted sites.

Preoperative intravenous rehydration for patients with pheochromocytomas and paragangliomas: is it necessary? A propensity score matching analysis.

BACKGROUND: Preoperative intravenous rehydration for patients with pheochromocytomas and paragangliomas (PPGLs) is widely used in many medical centers, but its usefulness has not been well evaluated. The objective of this study was to compare the perioperative hemodynamics and early outcome between patients who received preoperative intravenous rehydration and those without for resection of PPGLs. METHODS: In this retrospective propensity score-matched cohort study, the data of patients who underwent surgery for PPGLs were collected. Patients were divided into two groups depending on whether they received or did not receive intravenous rehydration preoperatively. The primary endpoint was intraoperative hypotension, described as the cumulative time of mean arterial pressure < 65 mmHg averaged by surgery duration. RESULTS: Among 231 enrolled patients, 113 patients received intravenous rehydration of ≥2000 ml daily for ≥2 days before surgery and 118 patients who did not have any intravenous rehydration before surgery. After propensity score matching, 85 patients remained in each group. The median cumulative time of mean arterial pressure < 65 mmHg averaged by surgery duration was not significantly different between rehydrated patients and non-rehydrated patients (median 3.0% [interquartile range 0.2-12.2] versus 3.8% [0.0-14.2], median difference 0.0, 95%CI - 1.2 to 0.8, p = 0.909). The total dose of catecholamines given intraoperatively, volume of intraoperative fluids, intraoperative tachycardia and hypertension, percentage of patients who suffered from postoperative hypotension, postoperative diuretics use, and postoperative early outcome between the two groups were not significantly different either. CONCLUSIONS: For patients with PPGLs, preoperative intravenous rehydration failed to optimize perioperative hemodynamics or improve early outcome.

[Characterization of AOX family members from Aquilaria sinensis and their responses to wounding].

Aquilaria sinensis is a typical inducible medicinal plant, that can produce agarwood only after it is wounded by external stimuli. Alternative oxidase(AOX) is one of the terminal oxidases of the plant mitochondrial electron transport, which plays an important role in plants' response to environmental stress. In order to reveal the physiological function of AOX gene in the process of agarwood formation from A.sinensis induced by wounding, AOX gene was cloned based on the transcriptome database and then identified by the bioinformatics analysis, and their expression pattern in different tissues and under wounding stress were detected by qRT-PCR. The results as follows. Three AOX genes were cloned from A.sinensis for the first time. They were named AsAOX1a, AsAOX1d and AsAOX2, respectively. The tissue expression shown that AsAOX1a is mainly expressed in the stem and the seed, and the AsAOX1d and AsAOX2 genes are mainly expressed in the pulp and the stem. AsAOX1a and AsAOX1d genes are highly responsive to wounding stress, and their response time was different. In addition, the expression of AsAOX1a and AsAOX2 induced by wounding are reduced by H_2O_2 treatment, but promoted by AsA treatment. The cloning, bioinformatics analysis and expression characteristics of AOX genes from A.sinensis provided basic information for further study the function of AOX genes in the development of A.sinensis, especially in the process of agarwood formation of A. sinensis induced by wounding.

Isolation and characterization of a novel cadmium-regulated Yellow Stripe-Like transporter (SnYSL3) in Solanum nigrum.

KEY MESSAGE: SnYSL3 encodes a plasma-localized transporter delivering various metal-nicotianamine complexes. The expression of SnYSL3 is up-regulated by excess Cd, suggesting an important role for SnYSL3 in response to Cd stress. The Yellow Stripe-Like (YSL) transporters have been proposed to participate in metal uptake and long-range transport in model plants. In this study, we isolated and characterized a novel member of the YSL gene family, SnYSL3, from the cadmium hyperaccumulator Solanum nigrum. SnYSL3 was constitutively expressed and encodes a plasma membrane-localized protein. In situ RNA hybridization localized the SnYSL3 transcripts predominantly in vascular tissues and epidermal cells of the roots and stems, while in leaves, the mRNA levels were high in the vasculature. The SnYSL3 expression level was up-regulated by excess Cd, excess Fe and Cu deficiency. Heterologous expression of SnYSL3 in yeast revealed that SnYSL3 transports nicotianamine complexes containing Fe(II), Cu, Zn and Cd. SnYSL3 overexpression in Arabidopsis thaliana decreased Fe and Mn concentrations in the roots and increased the root-to-shoot translocation ratios of Fe and Mn. Under Cd exposure, the transgenic plants showed increased translocation ratios of Fe and Cd, but no difference was observed in Mn translocation from roots to shoots between the transgenic and wild-type lines. Although the accurate function of SnYSL3 remains to be confirmed, these results suggest that SnYSL3 is a transporter delivering a broad range of metal-nicotianamine complexes and is potentially important for the response to heavy metal stress, especially due to Cd and Fe.

Quinoline-degrading strain Pseudomonas aeruginosa KDQ4 isolated from coking activated sludge is capable of the simultaneous removal of phenol in a dual substrate system.

Quinoline is a refractory organic compound in the treatment of coking wastewater. The isolation of high efficiency quinoline-degrading bacteria from activated sludge and the evaluation of their degradation characteristics in the presence of phenol or in the actual coking wastewater are important for the improvement of effluent quality. The novel bacterial strain Pseudomonas aeruginosa KDQ4 was isolated from a quinoline enrichment culture obtained from the activated sludge of a coking wastewater treatment plant. The optimum temperature and initial pH for quinoline degradation were 33-38°C and 8-9, respectively. KDQ4 completely degraded 400 mg/L of quinoline within 24 h and 800 mg/L of phenol within 30 h. In the dual-substrate system, the removal efficiencies of quinoline and phenol at the same initial concentration (200 mg/L) by KDQ4 were 89% and 100% within 24 h, respectively, indicating that KDQ4 could simultaneously and quickly degrade quinoline and phenol in a coexistence system. Moreover, KDQ4 was able to adapt to actual coking wastewater containing high quinoline and phenol concentrations and rapidly remove them. KDQ4 also exhibited heterotrophic nitrification and aerobic denitrification potential under aerobic conditions. These results suggested a potential bioaugmentation role for KDQ4 in the removal of nitrogen-heterocyclic compounds and phenolics from coking wastewater.

Identification of a cluster-situated activator of oxytetracycline biosynthesis and manipulation of its expression for improved oxytetracycline production in Streptomyces rimosus.

BACKGROUND: Oxytetracycline (OTC) is a broad-spectrum antibiotic commercially produced by Streptomyces rimosus. Despite its importance, little is known about the regulation of OTC biosynthesis, which hampered any effort to improve OTC production via engineering regulatory genes. RESULTS: A gene encoding a Streptomyces antibiotic regulatory protein (SARP) was discovered immediately adjacent to the otrB gene of oxy cluster in S. rimosus and designated otcR. Deletion and complementation of otcR abolished or restored OTC production, respectively, indicating that otcR encodes an essential activator of OTC biosynthesis. Then, the predicted consensus SARP-binding sequences were extracted from the promoter regions of oxy cluster. Transcriptional analysis in a heterologous GFP reporter system demonstrated that OtcR directly activated the transcription of five oxy promoters in E. coli, further mutational analysis of a SARP-binding sequence of oxyI promoter proved that OtcR directly interacted with the consensus repeats. Therefore, otcR was chosen as an engineering target, OTC production was significantly increased by overexpression of otcR as tandem copies each under the control of strong SF14 promoter. CONCLUSIONS: A SARP activator, OtcR, was identified in oxy cluster of S. rimosus; it was shown to directly activate five promoters from oxy cluster. Overexpression of otcR at an appropriate level dramatically increased OTC production by 6.49 times compared to the parental strain, thus demonstrating the great potential of manipulating OtcR to improve the yield of OTC production.

The N-terminal degenerated metal-binding domain is involved in the heavy metal transport activity of TaHMA2.

KEY MESSAGE: We identified key residues of TaHMA2, and the N- and C-terminal regions of the protein have different roles in its transport function when heterologously expressed in yeast. TaHMA2, a P1B-type ATPase from wheat (Triticum aestivum L.), plays an important role in heavy metal homeostasis in plants. A previous study showed that overexpressing TaHMA2 in rice (Oryza sativa L.), Arabidopsis thaliana, or tobacco (Nicotiana tabacum L.) resulted in various responses to heavy metals. Here, we report the heterologous expression of TaHMA2 in the yeast Saccharomyces cerevisiae. TaHMA2 expression increased the yeast's sensitivity to Cd, but not to Zn, Pb or Co, and increased Cd accumulation was concurrently observed. The eGFP-TaHMA2 fusion protein was localized to the plasma membrane and showed a discontinuous pattern. Mutagenesis of the cysteine and glutamate residues in the N-terminal metal-binding domain (N-MBD) impaired the function of TaHMA2. Deletion of most of the C terminus (TaHMA2ΔC, 712-1003) partially abolished the protein's function, whereas deletion of the N terminus (TaHMA2ΔN, 2-699) completely abolished Cd sensitivity. These data suggest that cysteine and glutamate residues are important for the metal-binding/translocation function of TaHMA2. Additional studies are needed to further understand the selectivity of TaHMA2 in planta.

EBV-Associated Smooth Muscle Tumors With Autoimmune Hemolytic Anemia and Hepatitis B Infection: Report of a Previously Undescribed Neoplasm With Review.

Epstein-Barr virus-associated smooth muscle tumor (EBV-SMT) is rare in adults. The presence of intratumoral T lymphocytes and primitive rounded cells characterized this neoplasm. We report a 24-year-old Chinese man who developed EBV-SMT in the right adrenal gland with hepatitis B infection and autoimmune hemolytic anemia without a history of HIV infection, primary immune deficiency, organ transplantation, or malignant tumor. This patient had an unknown immunodeficient state. EBV-SMTs are commonly located in the liver, lung, and gastrointestinal tract but rarely in the adrenal gland. We reviewed 10 reported literature on EBV-SMT in the adrenal gland. It is imperative to distinguish EBV-SMT from conventional somatic smooth muscle tumors. The discovery of EBV-SMT forces the clinician to conduct a thorough evaluation of immune function and immune status surveillance, and these patients are vulnerable to subsequent malignant tumors.

Co-immobilization of crosslinked enzyme aggregates on lysozyme functionalized magnetic nanoparticles for enhancing stability and activity.

Surface chemistry of carriers plays a key role in enzyme loading capacity, structure rigidity, and thus catalyze activity of immobilized enzymes. In this work, the two model enzymes of horseradish peroxidase (HRP) and glucose oxidase (GOx) are co-immobilized on the lysozyme functionalized magnetic core-shell nanocomposites (LYZ@MCSNCs) to enhance their stability and activity. Briefly, the HRP and GOx aggregates are firstly formed under the crosslinker of trimesic acid, in which the loading amount and the rigidity of the enzyme can be further increased. Additionally, LYZ easily forms a robust anti-biofouling nanofilm on the surface of SiO2@Fe3O4 magnetic nanoparticles with abundant functional groups, which facilitate chemical crosslinking of HRP and GOx aggregates with minimized inactivation. The immobilized enzyme of HRP-GOx@LYZ@MCSNCs exhibited excellent recovery activity (95.6 %) higher than that of the free enzyme (HRP&GOx). Specifically, 85 % of relative activity was retained after seven cycles, while 73.5 % of initial activity was also remained after storage for 33 days at 4 °C. The thermal stability and pH adaptability of HRP-GOx@LYZ@MCSNCs were better than those of free enzyme of HRP&GOx. This study provides a mild and ecofriendly strategy for multienzyme co-immobilization based on LYZ functionalized magnetic nanoparticles using HRP and GOx as model enzymes.

Dauricine Inhibits Non-small Cell Lung Cancer Development by Regulating PTEN/AKT/mTOR and Ras/MEK1/2/ERK1/2 Pathways in a FLT4-dependent Manner.

OBJECTIVE: Non-small cell lung cancer (NSCLC) is still a solid tumor with high malignancy and poor prognosis. Vascular endothelial growth factor receptor 3 (FLT4, VEGFR3) is overexpressed in NSCLC cells, making it a potential target for NSCLC treatment. In this study, we aimed to explore the anti-cancer effects of dauricine on NSCLC cells and its mechanism targeting FLT4. METHODS: We found that dauricine inhibited the growth of NCI-H1299 cells by blocking the cycle in the G2/M phase through flow cytometry analysis. In addition， dauricine also inhibited the migration of NCI-H1299 cells by wound healing assay and transwell migration assay. More importantly, our empirical analysis found the anti-cancer effect of dauricine on NCI-H1299 cells and the protein level of FLT4 had a distinctly positive correlation, and this effect was weakened after FLT4 knockdown. RESULTS: It is suggested that dauricine suppressed the growth and migration of NCI-H1299 cells by targeting FLT4. Furthermore, dauricine inhibited FLT4 downstream pathways, such as PTEN/AKT/mTOR and Ras/MEK1/2/ERK1/2, thereby regulating cell migration-related molecule MMP3 and cell cycle-related molecules (CDK1, pCDK1-T161, and cyclin B1). CONCLUSION: Dauricine may be a promising FLT4 inhibitor for the treatment of NSCLC.

HPLC-fluorescence detection for stability of harringtonine, and identification of degradation products by UPLC-Q-TOF-MS.

Harringtonine (HT) is an anticancer alkaloid early extracted and isolated from cephalotaxus fortunei Hook. f., also has various pharmacological activities such as antiviral, antibacterial, antimalarial, anti-inflammatory, antioxidant, herbicidal and insecticidal. However, the factors affecting the stability of HT, the main degradation sites and mechanisms involved in its disposal process in vivo have not yet been elucidated. This study utilized HPLC-fluorescence detection method to establish a simple quantitative detection method for HT with good accuracy, precision, and high sensitivity. Temperature and pH were the main factors affecting the stability of HT, which underwent significant degradation in high temperature and alkaline environments because of the occurrence of hydrolysis reactions. In isolated biological homogenates of SD rats, except gastrointestinal tract, HT was degraded in other sites, especially respiratory, mainly in airway and lungs, and systemic metabolism, mainly in livers, spleens, and kidneys. Through UPLC-Q-TOF-MS, three forced degradation products were identified as 4'-demethyl HT, cephalotaxine, and dehydrated HT, respectively. However, the degradation product in isolated biological homogenates of SD rats was only 4'-demethyl HT due to the relatively mild environment. Our findings contributed to a necessary study basis for HT in terms of structural optimization, dosage form selection, storage and transportation.

In vitro pharmacokinetic behavior in lung of harringtonine, an antagonist of SARS-CoV-2 associated proteins: New insights of inhalation therapy for COVID-19.

BACKGROUND: Recent studies have shown that harringtonine (HT) could specifically bind with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein and host cell transmembrane serine protease 2 (TMPRSS2) to block membrane fusion, which is an effective antagonist for SARS-CoV-2. PURPOSE: Our study focused on in-depth exploration of in vitro pharmacokinetic characteristics of HT in lung. METHODS: HPLC-fluorescence detection method was used to detect changes of HT content. Incubation systems of lung microsomes for phase I metabolism and UGT incubation systems for phase II metabolism were performed to elucidate metabolites and metabolic mechanisms of HT, and then the metabolic enzyme phenotypes for HT were clarified by chemical inhibition method and recombinant enzyme method. Through metabolomics, we comprehensively evaluated the physiological dynamic changes in SD rat and human lung microsomes, and revealed the relationship between metabolomics and pharmacological activity of HT. RESULTS: HPLC-fluorescence detection method showed strong specificity, high accuracy, and good stability for rapid quantification of HT. We confirmed that HT mainly underwent phase I metabolism, and the metabolites of HT in different species were all identified as 4'-demethyl HT, with metabolic pathway being hydrolysis reaction. CYP1A2 and CYP2E1 participated in HT metabolism, but as HT metabolism was not NADPH dependent, the esterase HCES1 in lung also played a role. The main KEGG pathways in SD rat and human lung microsomes were cortisol synthesis and secretion, steroid hormone biosynthesis and linoleic acid metabolism, respectively. The downregulated key biomarkers of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME suggested that HT could prevent immunosuppression and interfere with infection and replication of SARS-CoV-2. CONCLUSION: HT was mainly metabolized into 4'-demethyl HT through phase I reactions, which was mediated by CYP1A2, CYP2E1, and HCES1. The downregulation of 11-deoxycortisol, 21-deoxycortisol and 9(10)-EpOME were key ways of HT against SARS-CoV-2. Our study was of great significance for development and clinical application of HT in the treatment of COVID-19.