A strategy to distinguish similar traditional Chinese medicines by liquid chromatography-mass spectrometry, electronic senses, and gas chromatography-ion mobility spectrometry: Marsdeniae tenacissimae Caulis and Paederiae scandens Caulis as examples.

INTRODUCTION: Marsdeniae tenacissimae Caulis (MTC), a popular traditional Chinese medicine, has been widely used in the treatment of tumor diseases. Paederiae scandens Caulis (PSC), which is similar in appearance to MTC, is a common counterfeit product. It is difficult for traditional methods to effectively distinguish between MTC and PSC. Therefore, there is an urgent need for a rapid and accurate method to identify MTC and PSC. OBJECTIVES: The aim is to distinguish between MTC and PSC by analyzing the differences in nonvolatile organic compounds (NVOCs), taste, odor, and volatile organic compounds (VOCs). METHODS: Liquid chromatography-mass spectrometry (LC-MS) was utilized to analyze the NVOCs of MTC and PSC. Electronic tongue (E-tongue) and electronic nose (E-nose) were used to analyze their taste and odor respectively. Gas chromatography-ion mobility spectrometry (GC-IMS) was applied to analyze VOCs. Finally, multivariate statistical analyses were conducted to further investigate the differences between MTC and PSC, including principal component analysis, orthogonal partial least squares discriminant analysis, discriminant factor analysis, and soft independent modeling of class analysis. RESULTS: The results of this study indicate that the integrated strategy of LC-MS, E-tongue, E-nose, GC-IMS, and multivariate statistical analysis can be effectively applied to distinguish between MTC and PSC. Using LC-MS, 25 NVOCs were identified in MTC, while 18 NVOCs were identified in PSC. The major compounds in MTC are steroids, while the major compounds in PSC are iridoid glycosides. Similarly, the distinct taste difference between MTC and PSC was precisely revealed by the E-tongue. Specifically, the pronounced bitterness in PSC was proven to stem from iridoid glycosides, whereas the bitterness evident in MTC was intimately tied to steroids. The E-nose detected eight odor components in MTC and six in PSC, respectively. The subsequent statistical analysis uncovered notable differences in their odor profiles. GC-IMS provided a visual representation of the differences in VOCs between MTC and PSC. The results indicated a relatively high relative content of 82 VOCs in MTC, contrasted with 32 VOCs exhibiting a similarly high relative content in PSC. CONCLUSION: In this study, for the first time, the combined use of LC-MS, E-tongue, E-nose, GC-IMS, and multivariate statistical analysis has proven to be an effective method for distinguishing between MTC and PSC from multiple perspectives. This approach provides a valuable reference for the identification of other visually similar traditional Chinese medicines.

[Chemical composition analysis of Ganoderma lucidum based on UPLC-Orbitrap-HRMS and virtual screening of FXR activators for treatment of liver fibrosis].

The chemical composition of Ganoderma lucidum ethanol extracts was systematically analyzed and identified by ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry(UPLC-Orbitrap-HRMS). The fragmentation pattern of the representative chemical compounds was summarized, and the potential anti-liver fibrosis active compounds of G. lucidum acting on the farnesoid X receptor(FXR) target were studied to elucidate its pharmacodynamic substance basis. Preliminarily, 95 chemical constituents of G. lucidum ethanol extracts were identified, including 24 ganoderic acids, 9 ganoderenic acids, 13 lucidenic acids, 3 ganolucidic acids, 1 ganoderma lactone, 40 other triterpenoids, 4 fatty acids, and 1 other constituent. In addition, the fragmentation patterns of the representative compounds were also analyzed. The structural characteristics of ganoderic acids and ganoderenic acids were the C30 skeleton, containing free-COOH and-OH groups, which could easily lose H_2O and CO_2 to form fragment ions. The D-ring was mostly a five-membered ring, which was prone to breakage. Lucidenic acids were the lanosterolane-type of the C27 skeleton, and the side-chain structure became shorter and contained the same free-COOH and-OH compared with ganoderic acids, which had been reduced from 8 to 5 cartons and prone to lose H_2O and CO_2. Then, six reported FXR receptor agonists were selected to form a training set for establishing a pharmacophore model based on FXR ligands. The 95 identified chemical constituents of G. lucidum were matched with the pharmacophore, and the optimal pharmacophore model 02(sensitivity=0.750 00, specificity=0.555 56, ROC=0.750) was selected for the virtual screening of the G. lucidum compound library through the validation of the test set. Finally, 31 potential G. lucidum active constituents were screened and chosen to activate the FXRs. The ADMET results showed that ganoderic acid H and lucidenic acid J had less than 90% plasma protein binding rate and no hepatotoxicity, which could be used as FXR activators for developing clinical drugs for the treatment of liver fibrosis, either alone or in combination.

[Impact of chaperone-mediated autophagy on bilirubin-induced damage of mouse microglial cells]. / åˆ†å­ä¼´ä¾£ä»‹å¯¼çš„è‡ªå™¬å¯¹èƒ†çº¢ç´ è¯±å¯¼çš„å°é¼ å°èƒ¶è´¨ç»†èƒžæŸä¼¤çš„å½±å“.

OBJECTIVES: To investigate the effect of chaperone-mediated autophagy (CMA) on the damage of mouse microglial BV2 cells induce by unconjugated bilirubin (UCB). METHODS: The BV2 cell experiments were divided into two parts. (1) For the CMA activation experiment: control group (treated with an equal volume of dimethyl sulfoxide), QX77 group (treated with 20 µmol/L QX77 for 24 hours), UCB group (treated with 40 µmol/L UCB for 24 hours), and UCB+QX77 group (treated with both 20 µmol/L QX77 and 40 µmol/L UCB for 24 hours). (2) For the cell transfection experiment: LAMP2A silencing control group (treated with an equal volume of dimethyl sulfoxide), LAMP2A silencing control+UCB group (treated with 40 µmol/L UCB for 24 hours), LAMP2A silencing group (treated with an equal volume of dimethyl sulfoxide), and LAMP2A silencing+UCB group (treated with 40 µmol/L UCB for 24 hours). The cell viability was assessed using the modified MTT method. The expression levels of p65, nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), and cysteinyl aspartate specific proteinase-1 (caspase-1) were detected by Western blot. The relative mRNA expression levels of the inflammatory cytokines interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNF-α) were determined by real-time quantitative polymerase chain reaction. Levels of IL-6 and TNF-α in the cell culture supernatant were measured using ELISA. The co-localization of heat shock cognate protein 70 with p65 and NLRP3 was detected by immunofluorescence. RESULTS: Compared to the UCB group, the cell viability in the UCB+QX77 group increased, and the expression levels of inflammation-related proteins p65, NLRP3, and caspase-1, as well as the mRNA relative expression levels of IL-1ß, IL-6, and TNF-α and levels of IL-6 and TNF-α decreased (P<0.05). Compared to the control group, there was co-localization of heat shock cognate protein 70 with p65 and NLRP3 in both the UCB and UCB+QX77 groups. After silencing the LAMP2A gene, compared to the LAMP2A silencing control+UCB group, the LAMP2A silencing+UCB group showed increased expression levels of inflammation-related proteins p65, NLRP3, and caspase-1, as well as increased mRNA relative expression levels of IL-1ß, IL-6, and TNF-α and levels of IL-6 and TNF-α (P<0.05). CONCLUSIONS: CMA is inhibited in UCB-induced BV2 cell damage, and activating CMA may reduce p65 and NLRP3 protein levels, suppress inflammatory responses, and counteract bilirubin neurotoxicity.

Grape polysaccharides: compositional changes in grapes and wines, possible effects on wine organoleptic properties, and practical control during winemaking.

Polysaccharides present in grapes interact with wine sensory-active compounds (polyphenols and volatile compounds) via different mechanisms and can affect wine organoleptic qualities such as astringency, color and aroma. Studies on the role that grape polysaccharides play in wines are reviewed in this paper. First, the composition of grape polysaccharides and their changes during grape ripening, winemaking and aging are introduced. Second, different interaction mechanisms of grape polysaccharides and wine sensory-active compounds (flavanols, anthocyanins and volatiles) are introduced, and the possible effects on wine astringency, color and aroma caused by these interactions are illustrated. Finally, the control of the grape polysaccharide content in practice is discussed, including classical winemaking methods (applying different maceration enzymes, temperature control, co-fermentation, blending), modern vinification technologies (pulsed electric field, ultrasound treatment), and the development of new grape polysaccharide products.

Heel bone mineral density and various oral diseases: a bidirectional Mendelian randomization.

INTRODUCTION: Observational studies demonstrated that the relationship between bone mineral density and oral diseases is mixed. To access the association between heel bone mineral density and various oral diseases, we conducted the Mendelian randomization analysis to explore the association. MATERIALS AND METHODS: Two-sample bidirectional Mendelian analysis was used to explore the relationship between heel bone mineral density and various oral diseases. The inverse-variance weighted (IVW) was used as the primary effect estimate, and various methods were applied to test the reliability and stability of the results, namely MR-Egger, weighted median, simple mode, and weighted mode. RESULTS: This study showed that there was a negative relationship between heel BMD and periodontitis when heel BMD was used as an exposure factor and periodontitis as an outcome factor (IVW OR = 0.85; 95% CI, 0.75-0.95; p = 0.005). Bidirectional Mendelian randomization showed that there was no statistically significant association between periodontitis and heel bone mineral density when chronic periodontitis was the exposure factor (p > 0.05). And there was no significant relationship between heel bone mineral density and other oral diseases (dental caries, diseases of pulp and periapical tissues, impacted teeth, cleft lip, and cleft palate, oral and oropharyngeal cancer) (p > 0.05). CONCLUSION: This study showed that there was a negative relationship between heel bone density and periodontitis, and the decrease in heel bone density could promote the occurrence of periodontitis. In addition, there was no statistically significant relationship between heel bone density and other oral diseases.

Identification and Analysis of MADS-Box Genes Expressed in the Mesocarp of Oil Palm Fruit (Elaeis guineensis Jacq.).

Oil palm (Elaeis guineensis) is the most important tropical oil-bearing crop species worldwide. MADS-box proteins, which play crucial roles in plant growth and development and are involved in various physiological and biochemical processes, compose one of the largest families of plant transcription factors. In this study, 42 MADS-box genes were screened from the mesocarp transcriptome database of oil palm fruit, and their phylogenetic relationships with Arabidopsis thaliana MADS-box genes were analyzed. Based on the results, MADS-box genes from oil palm mesocarp were classified into four groups: MIKCc-type, MIKC*-type, Mα-type, and Mγ-type MADS-box genes. Members of the subfamilies were classified according to the presence of three specific protein motifs. To explore the differential expression of the MADS-box genes, the dynamic expression of all selected MADS-box genes in oil palm was measured by RNA-seq. The high expression of specific MADS-box genes in the mesocarp of oil palm during different developmental stages indicates that those genes may play important roles in the cell division of and metabolite accumulation in the fruit and could become important targets for fruit development and oil accumulation research in oil palm.

Morphology Deformation and Giant Electronic Band Modulation in Long-Wavelength WS2 Moiré Superlattices.

As a lattice interference effect, moiré superlattices feature a magnification effect that they respond sensitively to both the extrinsic mechanical perturbations and intrinsic atomic reconstructions. Here, using scanning tunneling microscopy and spectroscopy, we observe that long-wavelength WS2 superlattices are reconstructed into various moiré morphologies, ranging from regular hexagons to heavily deformed ones. We show that a dedicated interplay between the extrinsic nonuniform heterostrain and the intrinsic atomic reconstruction is responsible for this interesting moiré structure evolution. Importantly, the interplay between these two factors also introduces a local inhomogeneous intralayer strain within a moiré. Contrary to the commonly reported electronic modulation that occurred at the valence band edge due to interlayer hybridization, we find that this local intralayer strain induces a strong modulation at K point of the conduction band, reaching up to 300 meV in the heavily deformed moiré. Our microscopic explorations provide valuable information in understanding the intriguing physics in TMD moirés.

[Clinical observational study on the treatment of oligozoospermia with Shizi Sanhua decoction combined with Nuoyu].

OBJECTIVE: To study the clinical therapeutic effect as well as drug effectiveness and safety of Shizi Sanhua decoction combined with Nuoyu in the treatment of oligozoospermia in men. METHODS: 102 patients with oligozoospermia diagnosed at Longhua Hospital of Shanghai University of Traditional Chinese Medicine from February 2022 to March 2023 were selected and randomly divided into 3 groups. The treatment group was treated with Shizi Sanhua Decoction + Nuoyu; the traditional Chinese medicine group was treated with Shizi Sanhua Decoction; and the Nuoyu nutrient group was treated with Nuoyu nutrient. A review assessment and record were made after one course of treatment (3 months). RESULTS: A total of 102 patients completed the trial due to the treatment process. There were 34 cases in each of the traditional Chinese medicine group, the Nuoyu nutrient group, and the treatment group. Clinical efficacy: total effective rate of 52.94% in the traditional Chinese medicine group; 58.82% in the Nuoyu nutrient group; 82.35% in the treatment group. The clinical efficacy of the treatment group was better than that of the traditional Chinese medicine group and the Nuoyu nutrient group (P<0.05), which was statistically significant. Semen routine: the treatment group was better than the traditional Chinese medicine group and Nuoyu nutrient group in improving the total number of sperm and sperm concentration. CONCLUSION: The semen concentration and forward sperm count of patients with oligozoospermia treated with Shizi Sanhua Decoction combined with Nuoyu improved more significantly, and the clinical efficacy was remarkable. And the clinical efficacy is not affected by age and disease duration. It can be popularized and applied as a treatment for oligozoospermia.

[Correlation between active component content and color of Atractylodes Lancea and A. chinensis based on color difference principle].

We explored the correlations between the color difference values [ΔL~*(lightness), Δa~*(red-green), Δb~*(yellow-blue)] and the content of four active components(including sesquiterpenoids and polyacetylenes) in the powder of Atractylodes lancea and A. chinensis, aiming to provide reference for the quality evaluation of Atractylodis Rhizoma and establish a qualitative model that can distinguish between A. lancea and A. chinensis based on the chromatic values. The tristimulus values(L~*, a~*, and b~*) of 23 batches of A. lancea and A. chinensis were measured by a color difference meter. The content of atractylenolide Ⅱ, ß-eudesmol, atractylodin, and atractylone in the 23 batches of samples were measured by high performance liquid chromatography(HPLC). Principal component analysis(PCA) and partial least squares-discriminant analysis(PLS-DA) were performed to establish the qualitative models for distinguishing between A. lancea and A. chinensis. SPSS was employed to analyze the correlations between the tristimulus values and the content of the four index components. The results showed that the established PCA and PLS-DA models can divide the A. lancea and A. chinensis samples into two regions, and the tristimulus values of A. lancea and A. chinensis were positively correlated with the content of ß-eudesmol and atractylodin. Therefore, the PCA and PLS-DA models can successfully identify A. lancea and A. chinensis, and the appearance color can be used to quickly predict the internal quality of Atractylodis Rhizoma. This study provides a reference for the quality evaluation of Atractylodis Rhizoma and the modern research on the color of Chinese medicinal materials.

[Qualitative and quantitative study of flavonoids in Notoginseng Radix et Rhizoma based on UPLC-Q-TOF-MS and HPLC-DAD].

The flavonoids in Panax notoginseng were qualitatively analyzed by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF-MS), and the content of three main flavonoids in P. notoginseng of different specifications and grades collected from different habitats was determined by HPLC-DAD. Flavonoids and anthocyanins were analyzed by UPLC-Q-TOF-MS/MS in the positive and negative ion modes, respectively. Twelve flavonoid glycosides and one anthocyanin glycoside in P. notoginseng were identified, but no flavonoid aglycones were detected. Among them, 12 compounds were identified in the underground part of P. notoginseng for the first time and eight compounds were first reported in this plant. Moreover, six and four compounds were identified in the Panax genus and the Araliaceae family for the first time, respectively. A method for simultaneous determination of three flavonoids in P. notoginseng was established by HPLC-DAD. The content of flavonoids in 721 P. notoginseng samples of 124 specifications and grades collected from 20 different habitats was simultaneously determined. Among three flavonoids determined, the content of quercetin-3-O-(2″-ß-D-xylosyl)-ß-D-galactoside was the highest with the average content in the tested samples of 161.0 µg·g~(-1). The content of compounds quercetin-3-O-hexosyl-hexoside and kaempferol-3-O-pentosyl-hexoside was relatively low, with the average content of 18.5 µg·g~(-1)(calculated as quercetin-3-O-sophoroside) and 49.4 µg·g~(-1)(calculated as kaempferol-3-O-sangbu diglycoside). There were significant differences in flavonoids content of samples from different production area. The content of flavonoids in spring P. notoginseng was significantly lower than that in winter P. notoginseng when the other influencing factors such as production areas, germplasm resources, and cultivation conditions were fixed. As for P. notoginseng of different specifications, the flavonoid content in the part connecting the taproot and the aboveground stem was significantly higher than that in other parts. The results of large-scale data showed that the flavonoid content gradually increased with the increase in the number of heads. There were significant differences between the flavonoid content in most specifications and grades, especially the 20-head P. notoginseng and countless head P. notoginseng, whose content was significantly lower and significantly higher than that of other specifications and grades, respectively. This study provides a scientific basis for the study of the effective components and quality control of P. notoginseng from the perspective of flavonoids.

Integrated metabolomic and transcriptomic analysis reveals variation in the metabolites and genes of Platycodon grandiflorus roots from different regions.

INTRODUCTION: Platycodon grandiflorum root (PG), a popular traditional Chinese medicine, contains considerable chemical components with broad pharmacological activities. The complexity and diversity of the chemical components of PG from different origins contribute to its broad biological activities. The quality of southern PG is superior to that of northern PG, but the mechanisms underlying these differences remain unclear. OBJECTIVES: In order to study variation in the differentially accumulated metabolites (DAMs), differentially expressed genes (DEGs), as well as their interactions and signalling pathways among PG from Anhui and Liaoning. METHODS: The metabolomes based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the transcriptome based on high-throughput sequencing technology were combined to comprehensively analyse PGn and PGb. RESULTS: A total of 6515 DEGs and 83 DAMs from the comparison of PG from Anhui and Liaoning were detected. Integrated analysis of metabolomic and transcriptomic data revealed that 215 DEGs and 57 DAMs were significantly enriched in 48 pathways according to KEGG pathway enrichment analysis, and 15 DEGs and 10 DAMs significantly enriched in the main pathway sesquiterpenoid and triterpenoid and phenylpropanoid biosynthesis might play a key role in complex response or regulatory processes. CONCLUSION: Differences in PG from southern and northern China might thus stem from differences in environmental factors, such as precipitation, light duration, and humidity. The results of our study provide new insight into geographic variation in gene expression and metabolite accumulation and will enhance the utilisation of PG resources.

The effect of previous eradication failure on antibiotic resistance of Helicobacter pylori: A retrospective study over 8 years in Beijing.

BACKGROUND: Antibiotic resistance is the main cause of Helicobacter pylori (H. pylori) treatment failure. This study aimed to explore the characteristics of antibiotic resistance of H. pylori isolates in Beijing in the last 8 years and to estimate the impact of previous eradication failure on resistance patterns. MATERIALS AND METHODS: This retrospective study included data from a single center in Beijing from 2013 to 2020. Antibiotic susceptibility of 365 clinical H. pylori isolates was tested for amoxicillin, clarithromycin, metronidazole, levofloxacin, moxifloxacin, and tetracycline. The characteristics of the included patients and their previous eradication history were collected. Primary and secondary resistance rates of H. pylori to the six antibiotics and the impact of previous eradication failure on antibiotic resistance patterns were analyzed. RESULTS: The overall primary resistance rates of amoxicillin, clarithromycin, metronidazole, levofloxacin, moxifloxacin, and tetracycline were 0.7%, 55.2%, 68.0%, 49.7%, 64.5%, and 0%, with no significant increase during the observed period; while the secondary resistance rates were 3.2%, 96.7%, 90.7%, 93.1%, 80.0%, and 0%, respectively. The secondary resistance rate of clarithromycin (p < .001), metronidazole (p = .001), and levofloxacin (p < .001) significantly increased to 100% as the number of previous eradication therapies increased and exhibited a linear association. For strains naive to eradication, only 6.8% were susceptible to all the antibiotics, while 32.4% were single resistant, and 60.8% dual or multiple resistant. Clarithromycin+metronidazole+fluoroquinolone multiple resistance was the predominant pattern (0 course: 21.6%, 1 course: 37.5%, 2 courses: 56.1%, ≥3 courses: 71.1%; p < .001) for patients with treatment failure. The prevalence of dual or multiple-resistance patterns increased significantly as the number of previous therapies increased. CONCLUSIONS: The prevalence of primary and secondary resistance rates of clarithromycin, metronidazole, moxifloxacin, and levofloxacin were high in Beijing. Multiple-resistance patterns were common after treatment failure. Resistance rates of amoxicillin and tetracycline remained low and stable.

Valley Polarization and Inversion in Strained Graphene via Pseudo-Landau Levels, Valley Splitting of Real Landau Levels, and Confined States.

It is quite easy to control spin polarization and the spin direction of a system via magnetic fields. However, there is no such direct and efficient way to manipulate the valley pseudospin degree of freedom. Here, we demonstrate experimentally that it is possible to realize valley polarization and valley inversion in graphene by using both strain-induced pseudomagnetic fields and real magnetic fields. Pseudomagnetic fields, which are quite different from real magnetic fields, point in opposite directions at the two distinct valleys of graphene. Therefore, the coexistence of pseudomagnetic fields and real magnetic fields leads to imbalanced effective magnetic fields at two distinct valleys of graphene. This allows us to control the valley in graphene as conveniently as the electron spin. In this work, we report a consistent observation of valley polarization and inversion in strained graphene via pseudo-Landau levels, splitting of real Landau levels, and valley splitting of confined states using scanning tunneling spectroscopy. Our results highlight a pathway to valleytronics in strained graphene-based platforms.

Movable Valley Switch Driven by Berry Phase in Bilayer-Graphene Resonators.

Berry phase, the geometric phase accumulated over a closed loop in parameter space during an adiabatic cyclic evolution, has been demonstrated to play an important role in many quantum systems since its discovery. In gapped Bernal bilayer graphene, the Berry phase can be continuously tuned from zero to 2π, which offers a unique opportunity to explore the tunable Berry phase on physical phenomena. Here, we report experimental observation of Berry-phase-induced valley splitting and crossing in movable bilayer-graphene p-n junction resonators. In our experiment, the resonators are generated by combining the electric field of a scanning tunneling microscope tip with the gap of bilayer graphene. A perpendicular magnetic field changes the Berry phase of the confined bound states in the resonators from zero to 2π continuously and leads to the Berry phase difference for the two inequivalent valleys in the bilayer graphene. As a consequence, we observe giant valley splitting and unusual valley crossing of the lowest bound states. Our results indicate that the bilayer-graphene resonators can be used to manipulate the valley degree of freedom in valleytronics.

Tunable Lattice Reconstruction, Triangular Network of Chiral One-Dimensional States, and Bandwidth of Flat Bands in Magic Angle Twisted Bilayer Graphene.

The interplay between interlayer van der Waals interaction and intralayer lattice distortion can lead to structural reconstruction in slightly twisted bilayer graphene (TBG) with the twist angle being smaller than a characteristic angle θ_{c}. Experimentally, the θ_{c} is demonstrated to be very close to the magic angle (θ≈1.08°). Here we address the transition between reconstructed and unreconstructed structures of the TBG across the magic angle by using scanning tunneling microscopy (STM). Our experiment demonstrates that both structures are stable in the TBG around the magic angle. By using a STM tip, we show that the two structures can be changed to each other and a triangular network of chiral one-dimensional states hosted by domain boundaries can be switched on and off. Consequently, the bandwidth of the flat band, which plays a vital role in the emergent strongly correlated states in the magic angle TBG, is tuned. This provides an extra control knob to manipulate the exotic electronic states of the TBG near the magic angle.

A MADS-box gene, EgMADS21, negatively regulates EgDGAT2 expression and decreases polyunsaturated fatty acid accumulation in oil palm (Elaeis guineensis Jacq.).

KEY MESSAGE: EgMADS21 regulates PUFA accumulation in oil palm. Oil palm (Elaeis guineensis Jacq.) is the most productive world oil crop, accounting for 36% of world plant oil production. However, the molecular mechanism of the transcriptional regulation of fatty acid accumulation and lipid synthesis in the mesocarp of oil palm by up- or downregulating the expression of genes involved in related pathways remains largely unknown. Here, an oil palm MADS-box gene, EgMADS21, was screened in a yeast one-hybrid assay using the EgDGAT2 promoter sequence as bait. EgMADS21 is preferentially expressed in early mesocarp developmental stages in oil palm fruit and presents a negative correlation with EgDGAT2 expression. The direct binding of EgMADS21 to the EgDGAT2 promoter was confirmed by electrophoretic mobility shift assay. Subsequently, transient expression of EgMADS21 in oil palm protoplasts revealed that EgMADS21 not only binds to the EgDGAT2 promoter but also negatively regulates the expression of EgDGAT2. Furthermore, EgMADS21 was stably overexpressed in transgenic oil palm embryoids by Agrobacterium-mediated transformation. In three independent transgenic lines, EgDGAT2 expression was significantly suppressed by the expression of EgMADS21. The content of linoleic acid (C18:2) in the three transgenic embryoids was significantly decreased, while that of oleic acid (C18:1) was significantly increased. Combined with the substrate preference of EgDGAT2 identified in previous research, the results demonstrate the molecular mechanism by which EgMADS21 regulates EgDGAT2 expression and ultimately affects fatty acid accumulation in the mesocarp of oil palm.

Anti-Tumor Effects of Astaxanthin by Inhibition of the Expression of STAT3 in Prostate Cancer.

Astaxanthin is a natural product gaining increasing attention due to its safety and anti-cancer properties. In this study, we investigated the mechanisms of the anti-cancer effects of astaxanthin on prostate cancer (PCa) cell lines using aggressive PCa DU145 cells. Also an instantaneous silenced cell line (si-STAT3) derived from DU145 and a control cell line (si-NK) were used for the MTT and colony formation assays to determine the role of astaxanthin in proliferation and colony formation abilities. Flow cytometry assays were used to detect the apoptosis of tumor cells. Migration and invasion assays detected the weakening of the respective abilities. Western blot and RT-PCR tests detected the levels of STAT3 protein and mRNA. Astaxanthin resulted in suppression of the proliferation of DU145 cells and the level of STAT3. The treatment of DU145 cells with astaxanthin decreased the cloning ability, increased the apoptosis percentage and weakened the abilities of migration and invasion of the cells. Furthermore, astaxanthin reduced the expression of STAT3 at protein and mRNA levels. The effects were enhanced when astaxanthin and si-STAT3 were combined. The results of animal experiments were consistent with the results in cells. Thus, astaxanthin inhibits the proliferation of DU145 cells by reducing the expression of STAT3.

Study on Cecropin B2 Production via Construct Bearing Intein Oligopeptide Cleavage Variants.

In this study, genetic engineering was applied to the overexpression of the antimicrobial peptide (AMP) cecropin B2 (cecB2). pTWIN1 vector with a chitin-binding domain (CBD) and an auto-cleavage Ssp DnaB intein (INT) was coupled to the cecB2 to form a fusion protein construct and expressed via Escherichia coli ER2566. The cecB2 was obtained via the INT cleavage reaction, which was highly related to its adjacent amino acids. Three oligopeptide cleavage variants (OCVs), i.e., GRA, CRA, and SRA, were used as the inserts located at the C-terminus of the INT to facilitate the cleavage reaction. SRA showed the most efficient performance in accelerating the INT self-cleavage reaction. In addition, in order to treat the INT as a biocatalyst, a first-order rate equation was applied to fit the INT cleavage reaction. A possible inference was proposed for the INT cleavage promotion with varied OCVs using a molecular dynamics (MD) simulation. The production and purification via the CBD-INT-SRA-cecB2 fusion protein resulted in a cecB2 yield of 58.7 mg/L with antimicrobial activity.

[Role of pyroptosis in bilirubin-induced microglial injury].

OBJECTIVE: To study whether pyroptosis is involved in the bilirubin-induced injury of primary cultured rat cortical microglial cells. METHODS: Primary cultured rat cortical microglial cells were randomly administered with 30 µmol/L bilirubin (bilirubin group), 30 µmol/L bilirubin following 30 µmol/L VX-765 pretreatment (VX-765+bilirubin group), or an equal volume of dimethyl sulfoxide (control group). Modified MTT assay was used to measure the viability of microglial cells. Western blot was used to measure the expression of the pyroptosis-related proteins Caspase-1 and gasdermin D (GSDMD). Lactate dehydrogenase (LDH)-release assay was used to evaluate the cytotoxicity of microglial cells. EtBr/EthD2 with different molecular weights (394 Da/1 293 Da) was used to measure the size of plasma membrane pores. ELISA was used to measure the level of the inflammatory factor interleukin-1ß (IL-1ß) in culture supernatant. RESULTS: After bilirubin stimulation, the viability of microglial cells decreased and LDH release increased, both in a time-dependent manner. Compared with the control group, the bilirubin group had a significantly higher positive rate of small-molecule EtBr passing through the cell membrane (P<0.001), while there was no significant difference in the pass rate of large-molecule EthD2 between groups (P>0.05). The expression of activated Caspase-1 significantly increased at 0.5 hour after bilirubin stimulation (P<0.05), and that of activated GSDMD significantly increased at 6 hours after bilirubin stimulation (P<0.05). The release of IL-1ß significantly increased at 6 hours after bilirubin stimulation and reached the peak at 24 hours (P<0.001). Compared with the bilirubin group, the VX-765+bilirubin group had a significant increase in cell viability (P<0.05) and significant reductions in the expression of activated GSDMD, the pass rate of EtBr, and the release of LDH and IL-1ß (P<0.05). CONCLUSIONS: Pyroptosis is involved in bilirubin-induced injury of primary cultured microglial cells.

Comparative physiological, metabolomic, and transcriptomic analyses reveal developmental stage-dependent effects of cluster bagging on phenolic metabolism in Cabernet Sauvignon grape berries.

BACKGROUND: Light conditions significantly influence grape berry ripening and the accumulation of phenolic compounds, but the underlying molecular basis remains partially understood. Here, we applied integrated transcriptomics and pathway-level metabolomics analyses to investigate the effect of cluster bagging during various developmental stages on phenolic metabolism in Cabernet Sauvignon grapes. RESULTS: Bagging treatments had limited effects on berry quality attributes at harvest and did not consistently affect phenolic acid biosynthesis between seasons. Significantly elevated flavan-3-ol and flavonol contents were detected in re-exposed berries after bagging during early-developmental stages, while bagging after véraison markedly inhibited skin anthocyanin accumulation. Several anthocyanin derivatives and flavonol glycosides were identified as marker phenolic metabolites for distinguishing bagged and non-bagged grapes. Coordinated transcriptional changes in the light signaling components CRY2 and HY5/HYHs, transcription regulator MYBA1, and enzymes LAR, ANR, UFGT and FLS4, coincided well with light-responsive biosynthesis of the corresponding flavonoids. The activation of multiple hormone signaling pathways after both light exclusion and re-exposure treatments was inconsistent with the changes in phenolic accumulation, indicating a limited role of plant hormones in mediating light/darkness-regulated phenolic biosynthesis processes. Furthermore, gene-gene and gene-metabolite network analyses discovered that the light-responsive expression of genes encoding bHLH, MYB, WRKY, NAC, and MADS-box transcription factors, and proteins involved in genetic information processing and epigenetic regulation such as nucleosome assembly and histone acetylation, showed a high positive correlation with grape berry phenolic accumulation in response to different light regimes. CONCLUSIONS: Altogether, our findings provide novel insights into the understanding of berry phenolic biosynthesis under light/darkness and practical guidance for improving grape features.