Effects of Liqi Tongbian decoction on gut microbiota, SCFAs production, and 5-HT pathway in STC rats with Qi Stagnation Pattern.

Slow transit constipation (STC) is a common and debilitating condition characterized by delayed colonic transit and difficulty in fecal expulsion, significantly impacting patients' physical and mental wellbeing as well as their overall quality of life. This study investigates the therapeutic potential of Liqi Tongbian Decoction (LTD) in the treatment of STC, especially in cases involving the context of Qi stagnation, through a multifaceted approach involving the modulation of intestinal flora and short-chain fatty acids (SCFAs). We employed a rat model of STC with Qi Stagnation Pattern, established using the "loperamide + tail-clamping provocation method," to explore the effects of LTD on fecal characteristics, intestinal motility, and colonic pathology. Importantly, LTD exhibited the ability to increase the richness, diversity, and homogeneity of intestinal flora while also modulating the composition of microorganisms. It significantly increased the production of SCFAs, especially butyric acid. Moreover, LTD exerted a substantial influence on the synthesis of serotonin (5-HT) by modulating the expression of tryptophan hydroxylase (TPH) and interacting with the 5-HT4 receptor (5-HT4R), resulting in enhanced colonic motility. Correlation analyses revealed a positive correlation between certain bacterial genera, such as Lachnospiraceae_NK4A136 spp. and Clostridiales spp. and the concentrations of butyric acid and 5-HT. These results suggest a mechanistic link between microbiome composition, SCFAs production, and 5-HT synthesis. These findings highlight the potential of LTD to alleviate STC by facilitating a beneficial interplay among intestinal flora, SCFAs production, and 5-HT-mediated colonic motility, providing novel insights into the management of STC with Qi Stagnation Pattern.

CircGPC3 promotes hepatocellular carcinoma progression and metastasis by sponging miR-578 and regulating RAB7A/PSME3 expression.

CircRNAs are a class of highly stable noncoding RNAs that play an important role in the progression of many diseases, especially cancer. In this study, high-throughput sequencing was used to screen for abnormally expressed circRNAs, and we found that circGPC3 was overexpressed in HCC tissues. However, the underlying mechanism of circGPC3 in the development and metastasis of hepatocellular carcinoma (HCC) remains unknown. In our study, we found that circGPC3 was significantly upregulated in HCC tissues and cells and that its overexpression was positively correlated with overall survival, TNM stage and lymph node metastasis. In vivo and in vitro experiments showed that circGPC3 knockdown repressed HCC cell migration, invasion and proliferation and promoted apoptosis. Mechanistically, circGPC3 promoted HCC proliferation and metastasis through the miR-578/RAB7A/PSME3 axis. Our results demonstrate that circGPC3 contributes to the progression of HCC and provides an intervention target for HCC.

Characterization of the Cytochrome P450 CYP716C52 in Celastrol Biosynthesis and Its Applications in Engineered Saccharomyces cerevisiae.

Celastrol is a bioactive pentacyclic triterpenoid with promising therapeutic effects that is mainly distributed in Celastraceae plants. Although some enzymes involved in the celastrol biosynthesis pathway have been reported, many biosynthetic steps remain unknown. Herein, transcriptomics and metabolic profiles of multiple species in Celastraceae were integrated to screen for cytochrome P450s (CYPs) that are closely related to celastrol biosynthesis. The CYP716 enzyme, TwCYP716C52, was found to be able to oxidize the C-2 position of polpunonic acid, a precursor of celastrol, to form the wilforic acid C. RNAi-mediated repression of TwCYP716C52 in Tripterygium wilfordii suspension cells further confirmed its involvement in celastrol biosynthesis. The C-2 catalytic mechanisms of TwCYP716C52 were further explored by using molecular docking and site-directed mutagenesis experiments. Moreover, a modular optimization strategy was used to construct an engineered yeast to produce wilforic acid C at a titer of 5.8 mg·L-1. This study elucidates the celastrol biosynthetic pathway and provides important functional genes and sufficient precursors for further enzyme discovery.

Does Subjective Well-Being Improve Self-Rated Health from Undergraduate Studies to Three Years after Graduation in China?

The health status of emerging adults is at risk. Although subjective well-being is one of the factors closely associated with health, their longitudinal relationship is not clear among emerging adults. The study aimed to investigate the prospective relationship between self-rated health and subjective well-being in emerging adults. The study collected longitudinal data from a total of 1021 Chinese college students (537 males and 484 females) for five years, including two years in college and three years after graduation. In the baseline survey, the average age of the sample was 21.57 years old. Descriptive statistics indicated that both self-rated health and subjective well-being significantly decreased from the senior year of college to the year after graduation. Correlation analysis revealed that self-rated health and subjective well-being had a significant positive relationship. In the five-wave random intercept cross-lagged panel model, subjective well-being unidirectionally predicted self-rated health. In other words, the subjective well-being in the previous year could positively predict self-rated health in the following year, but the previous self-rated health could not predict subsequent subjective well-being. Given the significance of emerging adulthood to individual development, more attention and care should be dedicated to improving subjective well-being so as to maintain good health and engagement in work.

Structural and functional investigations of syn-copalyl diphosphate synthase from Oryza sativa.

The large superfamily of labdane-related diterpenoids is defined by the cyclization of linear geranylgeranyl pyrophosphate (GGPP), catalyzed by copalyl diphosphate synthases (CPSs) to form the basic decalin core, the copalyl diphosphates (CPPs). Three stereochemically distinct CPPs have been found in plants, namely (+)-CPP, ent-CPP and syn-CPP. Here, we used X-ray crystallography and cryo-EM methods to describe different oligomeric structures of a syn-copalyl diphosphate synthase from Oryza sativa (OsCyc1), and provided a cryo-EM structure of OsCyc1D367A mutant in complex with the substrate GGPP. Further analysis showed that tetramers are the dominant form of OsCyc1 in solution and are not necessary for enzyme activity in vitro. Through rational design, we identified an OsCyc1 mutant that can generate ent-CPP in addition to syn-CPP. Our work provides a structural and mechanistic basis for comparing different CPSs and paves the way for further enzyme design to obtain diterpene derivatives with specific chirality.

Structural and Catalytic Insight into the Unique Pentacyclic Triterpene Synthase TwOSC.

The oxidosqualene cyclase (OSC) catalyzed cyclization of the linear substrate (3S)-2,3-oxidosqualene to form diverse pentacyclic triterpenoid (PT) skeletons is one of the most complex reactions in nature. Friedelin has a unique PT skeleton involving a fascinating nine-step cation shuttle run (CSR) cascade rearrangement reaction, in which the carbocation formed at C2 moves to the other side of the skeleton, runs back to C3 to yield a friedelin cation, which is finally deprotonated. However, as crystal structure data of plant OSCs are lacking, it remains unknown why the CSR cascade reactions occur in friedelin biosynthesis, as does the exact catalytic mechanism of the CSR. In this study, we determined the first cryogenic electron microscopy structure of a plant OSC, friedelin synthase, from Tripterygium wilfordii Hook. f (TwOSC). We also performed quantum mechanics/molecular mechanics simulations to reveal the energy profile for the CSR cascade reaction and identify key residues crucial for PT skeleton formation. Furthermore, we semirationally designed two TwOSC mutants, which significantly improved the yields of friedelin and ß-amyrin, respectively.

The composition, pharmacological effects, related mechanisms and drug delivery of alkaloids from Corydalis yanhusuo.

Corydalis yanhusuo W. T. Wang, also known as yanhusuo, yuanhu, yanhu and xuanhu, is one of the herb components of many Chinese Traditional Medicine prescriptions such as Jin Ling Zi San and Yuanhu-Zhitong priscription. C. yanhusuo was traditionally used to relieve pain and motivate blood and Qi circulation. Now there has been growing interest in pharmacological effects of alkaloids, the main bioactive components of C. yanhusuo. Eighty-four alkaloids isolated from C. yanhusuo are its important bioactive components and can be characterized into protoberberine alkaloids, aporphine alkaloids, opiate alkaloids and others and proper extraction or co-administration methods modulate their contents and efficacy. Alkaloids from C. yanhusuo have various pharmacological effects on the nervous system, cardiovascular system, cancer and others through multiple molecular mechanisms such as modulating neurotransmitters, ion channels, gut microbiota, HPA axis and signaling pathways and are potential treatments for many diseases. Plenty of novel drug delivery methods such as autologous red blood cells, self-microemulsifying drug delivery systems, nanoparticles and others have also been investigated to better exert the effects of alkaloids from C. yanhusuo. This review summarized the alkaloid components of C. yanhusuo, their pharmacological effects and mechanisms, and methods of drug delivery to lay a foundation for future investigations.

Application of fluorescence endoscopy with methylene blue dye and indocyanine green dual-tracer method in sentinel lymph node biopsy for women with breast cancer.

Background: Indocyanine green (ICG) allows for the real-time visualization of lymphatic drainage and provides favorable performance for sentinel lymph node (SLN) mapping. However, the limited ability of tissue penetration of the near-infrared fluorescence of ICG may lead to the failure of lymph node detection in the traditional open approach of sentinel lymph node biopsy (SLNB) for breast cancer, especially in overweight or obese patients. To accurately and quickly detect SLNs, we applied fluorescence endoscopy with a dual-tracer method using ICG and methylene blue dye (MBD) in SLNB for breast cancer. We conducted this study to assess the feasibility and application value of this method in minimally invasive surgery. Methods: A total of 117 patients who received dual-tracer injection of ICG and MBD prior to endoscopic SLNB from November 2020 to September 2021 were examined in this study. The number of SLNs identified, the SLN identification rate, the time to identify the first SLN, the procedure duration, and the postoperative morbidity were analyzed. Results: Biopsied SLNs could be identified in 116 patients (99.15%) with an average number of 5.12±1.87 per patient. Blue-stained SLNs were found in 99 patients (84.62%) and fluorescent SLNs in 112 patients (95.73%). A total of 34 patients (29.06%) had positive SLNs. In 6 cases (5.13%), the positive SLNs were only stained with ICG fluorescence. In 1 case (0.85%), the positive SLNs were only blue-stained with no fluorescence staining. The mean durations for the identification of the first SLN and endoscopic SLNB were 7.14±6.31 and 37.75±16.94 min, respectively. Upper-limb lymphoedema was observed 5 cases (4.27%) during a median follow-up period of 10 months. Conclusions: The fluorescence endoscopy method assisted by dual tracer facilitates SLN detection with a comparatively short procedure duration and low complication rate. This approach could serve as a new method for SLNB for patients with breast cancer.

The role of AFAP1-AS1 in mitotic catastrophe and metastasis of triple-negative breast cancer cells by activating the PLK1 signaling pathway.

Triple-negative breast cancer (TNBC) is characterized by fast growth, high metastasis, high invasion, and a lack of therapeutic targets. Mitosis and metastasis of TNBC cells are two important biological behaviors in TNBC malignant progression. It is well known that the long noncoding RNA AFAP1-AS1 plays a crucial role in various tumors, but whether AFAP1-AS1 is involved in the mitosis of TNBC cells remains unknown. In this study, we investigated the functional mechanism of AFAP1-AS1 in targeting Polo-like Kinase 1 (PLK1) activation and participating in mitosis of TNBC cells. We detected the expression of AFAP1-AS1 in the TNBC patient cohort and primary cells by in situ hybridization (ISH), northern blot, fluorescent in situ hybridization (FISH) and cell nucleus/cytoplasm RNA fraction isolation. High AFAP1-AS1 expression was negatively correlated with overall survival (OS), disease-free survival (DFS), metastasis-free survival (MFS) and recurrence-free survival (RFS) in TNBC patients. We explored the function of AFAP1-AS1 by transwell, apoptosis, immunofluorescence (IF) and patient-derived xenograft (PDX) models in vitro and in vivo. We found that AFAP1-AS1 promoted TNBC primary cell survival by inhibiting mitotic catastrophe and increased TNBC primary cell growth, migration and invasion. Mechanistically, AFAP1-AS1 activated phosphorylation of the mitosis-associated kinase PLK1 protein. Elevated levels of AFAP1-AS1 in TNBC primary cells increased PLK1 pathway downstream gene expression, such as CDC25C, CDK1, BUB1 and TTK. More importantly, AFAP1-AS1 increased lung metastases in a mouse metastasis model. Taken together, AFAP1-AS1 functions as an oncogene that activates the PLK1 signaling pathway. AFAP1-AS1 could be used as a potential prognostic marker and therapeutic target for TNBC.

[Functional characterization and enzymatic properties of flavonoid glycosyltransferase gene CtUGT49 in Carthamus tinctorius].

Carthami Flos, as a traditional blood-activating and stasis-resolving drug, possesses anti-tumor, anti-inflammatory, and immunomodulatory pharmacological activities. Flavonoid glycosides are the main bioactive components in Carthamus tinctorius. Glycosyltransferase deserves to be studied in depth as a downstream modification enzyme in the biosynthesis of active glycoside compounds. This study reported a flavonoid glycosyltransferase CtUGT49 from C. tinctorius based on the transcriptome data, followed by bioinformatic analysis and the investigation of enzymatic properties. The open reading frame(ORF) of the gene was 1 416 bp, encoding 471 amino acid residues with the molecular weight of about 52 kDa. Phylogenetic analysis showed that CtUGT49 belonged to the UGT73 family. According to in vitro enzymatic results, CtUGT49 could catalyze naringenin chalcone to the prunin and choerospondin, and catalyze phloretin to phlorizin and trilobatin, exhibiting good substrate versatility. After the recombinant protein CtUGT49 was obtained by hetero-logous expression and purification, the enzymatic properties of CtUGT49 catalyzing the formation of prunin from naringenin chalcone were investigated. The results showed that the optimal pH value for CtUGT49 catalysis was 7.0, the optimal temperature was 37 ℃, and the highest substrate conversion rate was achieved after 8 h of reaction. The results of enzymatic kinetic parameters showed that the K_m value was 209.90 µmol·L~(-1) and k_(cat) was 48.36 s~(-1) calculated with the method of Michaelis-Menten plot. The discovery of the novel glycosyltransferase CtUGT49 is important for enriching the library of glycosylation tool enzymes and provides a basis for analyzing the glycosylation process of flavonoid glycosides in C. tinctorius.

Three-dimensional visual technique based on CT lymphography data combined with methylene blue in endoscopic sentinel lymph node biopsy for breast cancer.

BACKGROUND: The combined application of blue dye and radioisotopes is currently the primary mapping technique used for sentinel lymph node biopsy (SLNB) in breast cancer patients. However, radiocolloid techniques have not been widely adopted, especially in developing countries, given the strict restrictions on radioactive materials. Consequently, we carried out a retrospective study to evaluate the feasibility and accuracy of three-dimensional visualization technique (3DVT) based on computed tomography-lymphography (CT-LG) in endoscopic sentinel lymph node biopsy (ESLNB) for breast cancer. METHODS: From September 2018 to June 2020, 389 patients who underwent surgical treatment of breast cancer in our department were included in this study. The CT-LG data of these patients were reconstructed into digital 3D models and imported into Smart Vision Works V1.0 to locate the sentinel lymph node (SLN) and for visual simulation surgery. ESLNB and endoscopic axillary lymph node dissection were carried out based on this new technique; the accuracy and clinical value of 3DVT in ESLNB were analyzed. RESULTS: The reconstructed 3D models clearly displayed all the structures of breast and axilla, which favors the intraoperative detection of SLNs. The identification rate of biopsied SLNs was 100% (389/389). The accuracy, sensitivity, and false-negative rate were 93.83% (365/389), 93.43% (128/137), and 6.57% (9/137), respectively. Upper limb lymphedema occurred in one patient 3 months after surgery during the 12-month follow-up period. CONCLUSIONS: Our 3DVT based on CT-LG data combined with methylene blue in ESLNB ensures a high identification rate of SLNs with low false-negative rates. It, therefore, has the potential to serve as a new method for SLN biopsy in breast cancer cases.

Interaction between the Gut Microbiota and Intestinal Motility.

The gut microbiota is the largest symbiotic ecosystem with the host and has been proven to play an important role in maintaining the stability of the intestinal environment. The imbalance of the gut microbiota is caused by the imbalance between the symbiotic microbiota and the pathogenic microbiota. The commensal microbiome regulates intestinal motility, while the pathogenic microbiome causes intestinal motility disorder, resulting in disease development. Intestinal motility is a relatively general term, and its meaning may include intestinal muscle contraction, intestinal wall biomechanics, intestinal compliance, and transmission. The role of intestinal microecology and intestinal motility are interrelated, intestinal flora disorder mediates intestinal motility, and abnormal intestinal motility affects colonization of the intestinal flora. In this review, we briefly outlined the interaction between gut microbiota and intestinal motility and provided a reference for future studies.

Prospects and hot spots for mammalian target of rapamycin in the field of neuroscience from 2002 to 2021.

Mammalian target of rapamycin (mTOR) is an important molecule that regulates cell metabolism, growth, and proliferation in the nervous system. This study aimed to present the current study hot spots and predict the future development trend of the mTOR pathway in neurologic diseases using bibliometrics. We referred to the publications in the Web of Science Core Collection database. VOSviewer and CiteSpace programs were used to evaluate countries/regions, institutions, authors, journals, keywords, and citations showing the current study focus and predicting the future trend of mTOR in neuroscience. The search date ended on 19 June 2022, and there were 3,029 articles on mTOR in neuroscience from 2002 to 2021. Visual analysis showed that although the number of publications declined slightly in some years, the number of publications related to mTOR generally showed an upward trend, reaching its peak in 2021. It had the largest number of publications in the United States. Keywords and literature analysis showed that protein synthesis regulation, ischemia, mitochondrial dysfunction, oxidative stress, and neuroinflammation may be hot spots and future directions of the nervous system in mTOR studies. Recently, the most studied neurological diseases are Alzheimer's disease (AD), tuberous sclerosis complex (TSC), and depression, which are still worthy of further studies by researchers in the future. This can provide a useful reference for future researchers to study mTOR further in the field of neuroscience.

Biosynthesis, total synthesis, and pharmacological activities of aryltetralin-type lignan podophyllotoxin and its derivatives.

Covering: up to 2022Podophyllotoxin (PTOX, 1), a kind of aryltetralin-type lignan, was first discovered in the plant Podophyllum peltatum and its structure was clarified by W. Borsche and J. Niemann in 1932. Due to its potent anti-cancer and anti-viral activities, it is considered one of the molecules most likely to be developed into modern drugs. With the increasing market demand and insufficient storage of natural resources, it is crucial to expand the sources of PTOXs. The original extraction method from plants has gradually failed to meet the requirements, and the biosynthesis and total synthesis have become the forward-looking alternatives. As key enzymes in the biosynthetic pathway of PTOXs and their catalytic mechanisms being constantly revealed, it is possible to realize the heterogeneous biosynthesis of PTOXs in the future. Chemical and chemoenzymatic synthesis also provide schemes for strictly controlling the asymmetric configuration of the tetracyclic core. Currently, the pharmacological activities of some PTOX derivatives have been extensively studied, laying the foundation for clinical candidate drugs. This review focuses primarily on the latest research progress in the biosynthesis, total synthesis, and pharmacological activities of PTOX and its derivatives, providing a more comprehensive understanding of these widely used compounds and supporting the future search for clinical applications.

Key Glycosyltransferase Genes of Panax notoginseng: Identification and Engineering Yeast Construction of Rare Ginsenosides.

Panax notoginseng is one of the most famous valuable medical plants in China, and its broad application in clinical treatment has an inseparable relationship with the active molecules, ginsenosides. Ginsenosides are glycoside compounds that have varied structures for the diverse sugar chain. Although extensive work has been done, there are still unknown steps in the biosynthetic pathway of ginsenosides. Here, we screened candidate glycosyltransferase genes based on the previous genome and transcriptome data of P. notoginseng and cloned the full length of 27 UGT genes successfully. Among them, we found that PnUGT33 could catalyze different ginsenoside substrates to produce higher polarity rare ginsenosides by extending the sugar chain. We further analyzed the enzymatic kinetics and predicted the catalytic mechanism of PnUGT33 by simulating molecular docking. After that, we reconstructed the biosynthetic pathway of rare ginsenoside Rg3 and gypenoside LXXV in yeast. By combining the Golden Gate method and overexpressing the UDPG biosynthetic genes, we further improved the yield of engineering yeast strain. Finally, the shake-flask culture yield of Rg3 reached 51 mg/L and the fed-batch fermentation yield of gypenoside LXXV reached 94.5 mg/L, which was the first and highest record.

Longitudinal Relationship Between Self-Esteem and Academic Self-Efficacy Among College Students in China: Evidence From a Cross-Lagged Model.

The present study aimed to investigate the associations between self-esteem and academic self-efficacy among Chinese college students. Descriptive statistics showed that on average, students' academic self-efficacy experienced a downward trend in the first 3 years before rising slightly in the graduation year, and that male students had higher academic self-efficacy than females in the first 2 years, whereas female students' academic self-efficacy surpassed their male counterparts in the latter years. There were significant, positive associations between the two variables. With cross-lagged analysis, we found that students' self-esteem significantly predicted their subsequent academic self-efficacy from the freshman to the junior years, and the effects among male students endured longer and stronger. Implications of the findings were discussed.

Mechanistic analysis for the origin of diverse diterpenes in Tripterygium wilfordii.

Tripterygium wilfordii is a valuable medicinal plant rich in biologically active diterpenoids, but there are few studies on the origins of these diterpenoids in its secondary metabolism. Here, we identified three regions containing tandemly duplicated diterpene synthase genes on chromosomes (Chr) 17 and 21 of T. wilfordii and obtained 11 diterpene synthases with different functions. We further revealed that these diterpene synthases underwent duplication and rearrangement at approximately 2.3-23.7 million years ago (MYA) by whole-genome triplication (WGT), transposon mediation, and tandem duplication, followed by functional divergence. We first demonstrated that four key amino acids in the sequences of TwCPS3, TwCPS5, and TwCPS6 were altered during evolution, leading to their functional divergence and the formation of diterpene secondary metabolites. Then, we demonstrated that the functional divergence of three TwKSLs was driven by mutations in two key amino acids. Finally, we discovered the mechanisms of evolution and pseudogenization of miltiradiene synthases in T. wilfordii and elucidated that the new function in TwMS1/2 from the terpene synthase (TPS)-b subfamily was caused by progressive changes in multiple amino acids after the WGT event. Our results provide key evidence for the formation of diverse diterpenoids during the evolution of secondary metabolites in T. wilfordii.

Association of GSTM1, GSTT1, and GSTP1 Ile105Val polymorphisms with risk of age-related macular degeneration: a meta-analysis.

BACKGROUND: This study determined to evaluate the association between glutathione S-transferase (GST) polymorphisms, namely, GSTM1 (rs1183423000, presence/absence), GSTT1 (rs1601993659, presence/absence), and GSTP1 Ile105Val (rs1695, A>G) polymorphisms, and AMD risk. METHODS: We searched PubMed, Embase, and Web of Science databases from January 2000 to June 2021. The odds ratio (OR) and 95% confidence interval (95% CI) were used as effect sizes. Heterogeneity was assessed using the heterogeneity metric I2. RESULTS: Five relevant studies involving 875 patients with AMD and 966 healthy controls were included in this meta-analysis, four studies concerning GSTM1 null polymorphism, four studies regarding GSTT1 null polymorphism, and four studies on GSTP1 Ile105Val polymorphism. The GSTM1 null polymorphism, GSTT1 null polymorphism and GSTP1 Ile105Val polymorphism were not significantly associated with AMD risk (OR 1.13, 95% CI 0.73-1.75, p = 0.59; OR 1.05, 95% CI 0.81-1.36, p = 0.69; OR 1.20, 95% CI 0.97-1.47, p = 0.09, respectively). There was no association between the combined GSTM1 null genotype and GSTT1 null genotype and AMD risk (OR 1.16, 95% CI 0.42-3.17, p = 0.77). Subgroup analyses revealed that the GSTM1 null genotype was associated with an increased risk of AMD in the Turkish population (OR 1.67, 95% CI 1.13-2.47, p = 0.01) and the GSTM1 null genotype was associated with a decreased incidence of non-exudative AMD (OR 0.72, 95% CI 0.52-0.99, p = 0.01). There was no obvious risk of publication bias found. CONCLUSIONS: This meta-analysis indicated that there were no significant associations between GSTM1, GSTT1, and GSTP1 Ile105Val polymorphisms and AMD risk.

Jiawei Yanghe Decoction Regulates Bone-Lipid Balance through the BMP-SMAD Signaling Pathway to Promote Osteogenic Differentiation of Bone Mesenchymal Stem Cells.

Background: The Jiawei Yanghe decoction (JWYHD) is a traditional Chinese medicine formula for the treatment of osteoporosis, but its therapeutic mechanism has not been fully elucidated, and the therapeutic target of the intervention disease needs to be further verified. The dysfunction of bone mesenchymal stem cells (BMSCs) is considered to be an important pathogenesis of postmenopausal osteoporosis (PMOP). The purpose of this study was to explore how JWYHD regulates BMSC differentiation through the BMP-SMAD signal pathway. Methods: In the in vivo study, we used an ovariectomized PMOP rat (n = 36, 2-month-old, 200 ± 20 g) model and femur micro-CT analysis to study the effect of JWYHD on bone loss in rats. By immunofluorescence, the translocation expression of BMP2, a key protein in the pathway, was detected. Serum bone metabolism was detected by an enzyme-linked immunosorbent assay (ELISA). Alkaline phosphatase (ALP) activity was detected by alkaline phosphatase staining (ALPS), osteogenesis and matrix mineralization were detected by alizarin red staining (ARS), the adipogenic ability of BMSCs was detected by oil red staining (ORS), and CFU is used to detect the ability of cells to form colonies. The expression of related proteins was detected by western blotting. Results: In vivo and in vitro, the OP phenotypes of SD rats induced by ovariectomy (OVX) included impaired bone mineral density and microstructure, abnormal bone metabolism, and impaired MSC differentiation potential. JWYHD treatment reversed this trend and restored the differentiation potential of MSCs. JWYHD medicated serum and direct intervention of drugs activated the BMP-SMAD signaling pathway, promoted the osteogenic differentiation of BMSCs, and inhibited their adipogenic differentiation. Conclusions: Our data identified that JWYHD is an effective alternative drug for the treatment of PMOP that functions to stimulate the differentiation of BMSCs into osteoblasts in the BMP-SMAD signaling-dependent mechanism.

Metabolic Engineering of Saccharomyces cerevisiae for High-Level Friedelin via Genetic Manipulation.

Friedelin, the most rearranged pentacyclic triterpene, also exhibits remarkable pharmacological and anti-insect activities. In particular, celastrol with friedelin as the skeleton, which is derived from the medicinal plant Tripterygium wilfordii, is a promising drug due to its anticancer and antiobesity activities. Although a previous study achieved friedelin production using engineered Saccharomyces cerevisiae, strains capable of producing high-level friedelin have not been stably engineered. In this study, a combined strategy was employed with integration of endogenous pathway genes into the genome and knockout of inhibiting genes by CRISPR/Cas9 technology, which successfully engineered multiple strains. After introducing an efficient TwOSC1T502E, all strains with genetic integration (tHMG1, ERG1, ERG20, ERG9, POS5, or UPC2.1) showed a 3.0∼6.8-fold increase in friedelin production compared with strain BY4741. Through further double knockout of inhibiting genes, only strains GD1 and GD3 produced higher yields. Moreover, strains GQ1 and GQ3 with quadruple mutants (bts1; rox1; ypl062w; yjl064w) displayed similar increases. Finally, the dominant strain GQ1 with TwOSC1T502E was cultured in an optimized medium in shake flasks, and the final yield of friedelin reached 63.91 ± 2.45 mg/L, which was approximately 65-fold higher than that of the wild-type strain BY4741 and 229% higher than that in ordinary SD-His-Ura medium. It was the highest titer for friedelin production to date. Our work provides a good example for triterpenoid production in microbial cell factories and lays a solid foundation for the mining, pathway analysis, and efficient production of valuable triterpenoids with friedelin as the skeleton.