ObjectiveBased on traditional quality evaluation of Gardeniae Fructus（GF） recorded in historical materia medica， this study systematically compared the quality differences between wild and cultivated GF from morphological characteristics， microscopic features， and contents of primary and secondary metabolites. MethodsVernier calipers and analytical balances were used to measure the length， diameter and individual fruit weight of wild and cultivated GF， and the aspect ratio was calculated. A colorimeter was used to determine the chromaticity value of wild and cultivated GF， and the paraffin sections of them were prepared by safranin-fast green staining and examined under an optical microscope to observe their microstructure. Subsequently， the contents of water-soluble and alcohol-soluble extracts of wild and cultivated GF were detected by hot immersion method under the general rule 2201 in volume Ⅳ of the 2020 edition of the Pharmacopoeia of the People's Republic of China， the starch content was measured by anthrone colorimetric method， the content of total polysaccharides was determined by phenol-sulfuric acid colorimetric method， the sucrose content was determined by high performance liquid chromatography coupled with evaporative light scattering detection（HPLC-ELSD）， and the contents of representative components in them were measured by ultra-performance liquid chromatography（UPLC）. Finally， correlation analysis was conducted between quality traits and phenotypic traits， combined with multivariate statistical analysis methods such as principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA）， key differential components between wild and cultivated GF were screened. ResultsIn terms of traits， the wild GF fruits were smaller， exhibiting reddish yellow or brownish red hues with significant variation between batches. While the cultivated GF fruits are larger， displaying deeper orange-red or brownish red. The diameter and individual fruit weight of cultivated GF were significantly greater than those of wild GF， while the blue-yellow value（b^*） of wild GF was significantly higher than that of cultivated GF. In the microstructure， the mesocarp of wild GF contained numerous scattered calcium oxalate cluster crystals， while the endocarp contained stone cell class round， polygonal or tangential prolongation， undeveloped seeds were visible within the fruit. In contrast， the mesocarp of cultivated GF contained few calcium oxalate cluster crystals， or some batches exhibited extremely numerous cluster crystals. The stone cells in the endocarp were predominantly round-like， with the innermost layer arranged in a grid pattern. Seeds were basically mature， and only a few immature seeds existed in some batches. Regarding primary metabolite content， wild GF exhibited significantly higher total polysaccharide level than cultivated GF（P<0.01）. In category-specific component content， wild GF exhibited significantly higher levels of total flavonoids and total polyphenols compared to cultivated GF（P<0.01）. Analysis of 12 secondary metabolites revealed that wild GF exhibited significantly higher levels of Shanzhiside， deacetyl asperulosidic acid methyl ester， gardenoside and chlorogenic acid compared to cultivated GF（P<0.01）. Conversely， the contents of genipin 1-gentiobioside， geniposide and genipin were significantly lower in wild GF（P<0.01）. ConclusionThere are significant differences between wild and cultivated GF in terms of traits， microstructure， and contents of primary and secondary metabolites. At present， the quality evaluation system of cultivated GF remains incomplete， and this study provides a reference for guiding the production of high-quality GF medicinal materials.

Diterpenoid ferruginol is a key intermediate in biosynthesis of active ingredients such as tanshinone and carnosic acid.However, the traditional process of obtaining ferruginol from plants is often cumbersome and inefficient. In recent years, the increasingly developing gene editing technology has been gradually applied to the heterologous production of natural products, but the production of ferruginol in microbe is still very low, which has become an obstacle to the efficient biosynthesis of downstream chemicals, such as tanshinone. In this study, miltiradiene was produced by integrating the shortened diterpene synthase fusion protein,and the key genes in the MVA pathway were overexpressed to improve the yield of miltiradiene. Under the shake flask fermentation condition, the yield of miltiradiene reached about(113. 12±17. 4)mg·L~(-1). Subsequently, this study integrated the ferruginol synthase Sm CYP76AH1 and Sm CPR1 to reconstruct the ferruginol pathway and thereby realized the heterologous synthesis of ferruginol in Saccharomyces cerevisiae. The study selected the best ferruginol synthase(Il CYP76AH46) from different plants and optimized the expression of pathway genes through redox partner engineering to increase the yield of ferruginol. By increasing the copy number of diterpene synthase, CYP450, and CPR, the yield of ferruginol reached(370. 39± 21. 65) mg·L~(-1) in the shake flask, which was increased by 21. 57-fold compared with that when the initial ferruginol strain JMLT05 was used. Finally, 1 083. 51 mg·L~(-1) ferruginol was obtained by fed-batch fermentation, which is the highest yield of ferruginol from biosynthesis so far. This study provides not only research ideas for other metabolic engineering but also a platform for the construction of cell factories for downstream products.
Saccharomyces cerevisiae/genetics* ; Diterpenes/metabolism* ; Metabolic Engineering ; Fermentation ; Abietanes

Based on the interleukin-17(IL-17) signaling pathway, this study explores the effect and mechanism of Bufei Decoction on Klebsiella pneumoniae pneumonia in rats. SD rats were randomly divided into the control group, model group, Bufei Decoction low-dose group(6.68 g·kg~(-1)·d~(-1)), Bufei Decoction high-dose group(13.36 g·kg~(-1)·d~(-1)), and dexamethasone group(1.04 mg·kg~(-1)·d~(-1)), with 10 rats in each group. A pneumonia model was established by tracheal drip injection of K. pneumoniae. After successful model establishment, the improvement in lung tissue damage was observed following drug administration. Core targets and signaling pathways were screened using transcriptomics techniques. Real-time fluorescence quantitative polymerase chain reaction was used to detect the mRNA expression of core targets interleukin-6(IL-6), interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and chemokine CXC ligand 6(CXCL6). Western blot was used to assess key proteins in the IL-17 signaling pathway, including interleukin-17A(IL-17A), nuclear transcription factor-κB activator 1(Act1), tumor necrosis factor receptor-associated factor 6(TRAF6), and downstream phosphorylated p38 mitogen-activated protein kinase(p-p38 MAPK), and phosphorylated nuclear factor-κB p65(p-NF-κB p65). Apoptosis of lung tissue cells was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling(TUNEL). The results showed that, compared with the control group, the model group exhibited significant pathological damage in lung tissue. The mRNA expression of IL-6, IL-1β, TNF-α, and CXCL6, as well as the protein levels of IL-17A, Act1, TRAF6, p-p38 MAPK/p38 MAPK, and p-NF-κB p65/NF-κB p65, were significantly increased, and the number of apoptotic cells was notably higher, indicating successful model establishment. Compared with the model group, both low-and high-dose groups of Bufei Decoction showed reduced pathological damage in lung tissue. The mRNA expression levels of IL-6, IL-1β, TNF-α, and CXCL6, and the protein levels of IL-17A, Act1, TRAF6, p-p38 MAPK/p38 MAPK, and p-NF-κB p65/NF-κB p65, were significantly decreased, with a significant reduction in apoptotic cells in the high-dose group. In conclusion, Bufei Decoction can effectively improve lung tissue damage and reduce inflammation in rats with K. pneumoniae. The mechanism may involve the regulation of the IL-17 signaling pathway and the reduction of apoptosis.
Animals ; Interleukin-17/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Rats, Sprague-Dawley ; Signal Transduction/drug effects* ; Rats ; Male ; Klebsiella pneumoniae/physiology* ; Klebsiella Infections/immunology* ; Humans ; Lung/drug effects*

OBJECTIVES: To report the development, validation, and findings of the Multi-dimensional Attention Rating Scale (MARS), a self-report tool crafted to evaluate six-dimension attention levels. METHODS: The MARS was developed based on Classical Test Theory (CTT). Totally 202 highly educated healthy adult participants were recruited for reliability and validity tests. Reliability was measured using Cronbach's alpha and test-retest reliability. Structural validity was explored using principal component analysis. Criterion validity was analyzed by correlating MARS scores with the Toronto Hospital Alertness Test (THAT), the Attentional Control Scale (ACS), and the Attention Network Test (ANT). RESULTS: The MARS comprises 12 items spanning six distinct dimensions of attention: focused attention, sustained attention, shifting attention, selective attention, divided attention, and response inhibition.As assessed by six experts, the content validation index (CVI) was 0.95, the Cronbach's alpha for the MARS was 0.78, and the test-retest reliability was 0.81. Four factors were identified (cumulative variance contribution rate 68.79%). The total score of MARS was correlated positively with THAT (r = 0.60, P < 0.01) and ACS (r = 0.78, P < 0.01) and negatively with ANT's reaction time for alerting (r = -0.31, P = 0.049). CONCLUSIONS The MARS can reliably and validly assess six-dimension attention levels in real-world settings and is expected to be a new tool for assessing multi-dimensional attention impairments in different mental disorders.
Humans ; Adult ; Male ; Attention/physiology* ; Female ; Middle Aged ; Reproducibility of Results ; Young Adult ; Psychometrics

Apical microsurgery is accurate and minimally invasive, produces few complications, and has a success rate of more than 90%. However, due to the lack of awareness and understanding of apical microsurgery by dental general practitioners and even endodontists, many clinical problems remain to be overcome. The consensus has gathered well-known domestic experts to hold a series of special discussions and reached the consensus. This document specifies the indications, contraindications, preoperative preparations, operational procedures, complication prevention measures, and efficacy evaluation of apical microsurgery and is applicable to dentists who perform apical microsurgery after systematic training.
Microsurgery/standards* ; Humans ; Apicoectomy ; Contraindications, Procedure ; Tooth Apex/diagnostic imaging* ; Postoperative Complications/prevention & control* ; Consensus ; Treatment Outcome

Objective To verify the efficacy of a domestic human DNA quantification kit based on real-time fluorescence quantitative PCR in detecting the total human DNA concentration,male DNA concen-tration in mixed male/female DNA samples,the degree of DNA degradation and inhibitor tolerance.Methods Samples with different concentrations,different male/female ratios,different concentrations of inhibitors,and different degradation degrees were tested using the domestic human DNA quantification kit based on real-time fluorescence quantitative PCR.This kit was compared with a similar product on the market and was applied to the detection of DNA from real cases.Results This human DNA quan-tification kit can effectively detect human DNA as low as 0.001 65 ng/μL,and 6.25 pg/μL of male DNA in mixed samples with a male-to-female ratio of 1∶15 000.Even when the sample contains as high as 400 ng/μL of humic acid or 1 000 μmol/L of hemin alone,the DNA concentration can still be accurately detected.The degradation index can effectively characterize the degradation degree of the sample.This kit has been successfully applied in forensic practice.Conclusion This human DNA quan-tification kit is accurate and reliable in detection.It can accurately reflect the degradation of DNA and inhibitor tolerance.It has good performance in quantitative accuracy,determination of the male/female ratio in mixed samples,and inhibitor tolerance.It has application potential in forensic case examination.

Aim To investigate the effect of p-AMPK activity on autophagy in different subtypes of MDA-MB-231(triple-negative breast cancer cells)and MCF-7(estrogen receptor-positive cells)and its regulatory mechanism.Methods MDA-MB-231 cells were trea-ted with EBSS,Baf-A1,and EBSS+Baf-A1 for four hours,and MCF-7 cells for eight hours.The effects of autophagy on cell proliferation and apoptosis were ob-served,mitochondrial morphology was examined,and the expression of autophagy markers LC3B,P62,LAMP1,TOM20,AMPK,p-AMPK,ULK1,and Bec-lin1/VPS34 proteins was detected.The autophagy pathway was validated by inhibiting AMPK activity.Results Breast cancer cells underwent autophagy af-ter starvation induction(EBSS),with inconsistent au-tophagy processes observed in different subtypes of breast cancer cells.Autophagy inhibited cell prolifera-tion.In MDA-MB-231 cells,autophagy led to an in-crease in p-AMPK levels and a decrease in ULK1 lev-els,initiating autophagy through p-AMPK activation of ULK1.In MCF-7 cells,both p-AMPK and ULK1 levels decreased after autophagy,suggesting that autophagy might not be mediated by p-AMPK activation.Conclu-sions MDA-MB-231 cells primarily initiate autophagy by directly activating ULK1 by p-AMPK,independent of the MTOR pathway.In MCF-7 cells autophagy might be triggered by inhibiting MTOR through AMPK activity or directly activating MTOR through other up-stream factors.Regulating p-AMPK activity based on the autophagy pathways in different cell subtypes could enable more precise targeting and treatment of different types of breast cancer.

Aim To investigate the effect of p-AMPK activity on autophagy in different subtypes of MDA-MB-231(triple-negative breast cancer cells)and MCF-7(estrogen receptor-positive cells)and its regulatory mechanism.Methods MDA-MB-231 cells were trea-ted with EBSS,Baf-A1,and EBSS+Baf-A1 for four hours,and MCF-7 cells for eight hours.The effects of autophagy on cell proliferation and apoptosis were ob-served,mitochondrial morphology was examined,and the expression of autophagy markers LC3B,P62,LAMP1,TOM20,AMPK,p-AMPK,ULK1,and Bec-lin1/VPS34 proteins was detected.The autophagy pathway was validated by inhibiting AMPK activity.Results Breast cancer cells underwent autophagy af-ter starvation induction(EBSS),with inconsistent au-tophagy processes observed in different subtypes of breast cancer cells.Autophagy inhibited cell prolifera-tion.In MDA-MB-231 cells,autophagy led to an in-crease in p-AMPK levels and a decrease in ULK1 lev-els,initiating autophagy through p-AMPK activation of ULK1.In MCF-7 cells,both p-AMPK and ULK1 levels decreased after autophagy,suggesting that autophagy might not be mediated by p-AMPK activation.Conclu-sions MDA-MB-231 cells primarily initiate autophagy by directly activating ULK1 by p-AMPK,independent of the MTOR pathway.In MCF-7 cells autophagy might be triggered by inhibiting MTOR through AMPK activity or directly activating MTOR through other up-stream factors.Regulating p-AMPK activity based on the autophagy pathways in different cell subtypes could enable more precise targeting and treatment of different types of breast cancer.

Tripterifordin and neotripterifordin are important ent-kaurane diterpenoids in the Chinese medicinal herb Tripterygium wilfordii, possessing significant anti-HIV(human immunodeficiency virus) activity. On the basis of elucidating the natural biosynthetic pathways of these compounds, heterologous production with microbial cell factories can help to alleviate the reliance on plant resources and provide abundant raw materials for sustainable production. TwKO is the first CYP450 enzyme involved in the biosynthesis of tripterifordin and neotripterifordin. This study aimed to enhance the catalytic activity of TwKO by site-directed mutagenesis to benefit the production of tripterifordin and neotripterifordin in yeast. The AlphaFold DB established based on the AlphaFold 2 was employed to obtain the protein model of TwKO. According to multiple sequence alignments and principles of natural evolution, the key residues influencing the binding of TwKO to the substrate were identified. Subsequently, functional characterization of the mutants were conducted in Saccharomyces cerevisiae. A total of 71 mutants were obtained, among which 11 and 11 mutants had the abilities of enhancing the production of 16α-hydroxy-ent-kaurenol and 16α-hydroxy-ent-kaurenoic acid, respectively. In addition, 10 mutants could increase the proportion of the oxidation product of 16α-hydroxy-ent-kaurenol. In particular, R304 was identified as a key residue affecting the catalytic specificity of TwKO, the mutation of which led to the specific prodiction of 16α-hydroxy-ent-kaurenol. This study was the first to reveal the key residue affecting the catalytic activity of TwKO and obtained the mutants with increased TwKO activity, lay a foundation for the biosynthesis of tripterifordin and neotripterifordin.
Tripterygium/chemistry* ; Mutagenesis, Site-Directed ; Diterpenes, Kaurane/chemistry* ; Plant Proteins/chemistry* ; Cytochrome P-450 Enzyme System/chemistry* ; Saccharomyces cerevisiae/metabolism*

Objective: To compare the clinical outcomes and biomechanical characteristics of 1-, 2-, and 3-level pedicle subtraction osteotomy (PSO), and establish selection criteria based on preoperative radiographic parameters. Methods: Patients undergone PSO to treat ankylosing spondylitis from February 2009 to May 2019 in Sun Yat-sen Memorial Hospital of Sun Yat-sen University were enrolled. According to the quantity of osteotomy performed, the participants were divided into group A (1-level PSO, n = 24), group B (2-level PSO, n = 19), and group C (3-level PSO, n = 11). Clinical outcomes were assessed before surgery and at the final follow-up. Comparisons of the radiographic parameters and quality-of-life indicators were performed among and within these groups, and the selection criteria were established by regression. Finite element analysis was conducted to compare the biomechanical characteristics of the spine treated with different quantity of osteotomies under different working conditions. Results: Three-level PSO improved the sagittal parameters more significantly, but resulted in longer operative time and greater blood loss (p < 0.05). Greater stress was found in the proximal screws and proximal junction area of the vertebra in the model simulating 1-level PSO. Larger stress of screws and vertebra was observed at the distal end in the model simulating 3-level PSO. Conclusion Multilevel PSO works better for larger deformity correction than single-level PSO by allowing greater sagittal parameter correction and obtaining a better distribution of stress in the hardware construct, although with longer operation time and greater blood loss. Three-level osteotomy is recommended for the patients with preoperative of global kyphosis > 85.95°, T1 pelvic angle > 62.3°, sagittal vertical alignment > 299.55 mm, and pelvic tilt+ chin-brow vertical angle > 109.6°.