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

Buyang Huanwu Decoction(BYHWD), as one of the classic formulas in traditional Chinese medicine(TCM) for the treatment of cerebral ischemic stroke(CIS), has demonstrated definite effects in clinical practice. However, the material basis and mechanism of treatment have not been systematically elucidated. This study employed network pharmacology and molecular docking to analyze the potential targets and mechanisms of blood-and brain-penetrating active components of BYHWD in reducing cell apoptosis in CIS. Cell experiments were then carried out to validate the prediction results. In the experiments, five active components including hydroxysafflor yellow A( HSYA), tetramethylpyrazine( TMP), astragaloside Ⅳ( AS-Ⅳ), amygdalin( AMY), and paeoniflorin(PF) were selected to explore the pharmacological effects of BYHWD. HT22 cells were treated with BYHWD, and the cell counting kit-8(CCK-8) method was employed to examine the toxic and side effects of BYHWD. A cell model of oxygen-glucose deprivation/reoxygenation( OGD/R) was constructed, with apoptosis and pyroptosis as the main screening indicators. The levels of lactate dehydrogenase(LDH) and glutathione(GSH) were measured to assess the cell membrane integrity. Flow cytometry was employed to detect apoptosis, and the activities of caspase-3 and caspase-1 were measured to clarify the status of apoptosis and pyroptosis. ELISA was employed to determine the levels of interleukin(IL)-1β and IL-18 to confirm pyroptosis. HSYA and AMY were identified in this study as the active components regulating apoptosis and pyroptosis. TUNEL was employed to detect the apoptosis rate, and Western blot was employed to determine the expression levels of apoptosis-related proteins B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), and caspase-3, which confirmed that the anti-apoptotic effect of the combined component group was superior to that of the single component groups. The molecular docking results revealed strong binding affinity of HSYA and AMY with SDF-1α and CXCR4.AMD3100, a selective antagonist of CXCR4, was then used for intervention. The results of Western blot showed alterations in the expression levels of apoptosis-associated proteins, SDF-1α, and CXCR4. In conclusion, HSYA and AMY influence cellular apoptosis by modulating the SDF-1α/CXCR4 signaling cascade.
Drugs, Chinese Herbal/chemistry* ; Apoptosis/drug effects* ; Animals ; Neurons/cytology* ; Mice ; Molecular Docking Simulation ; Cell Line ; Glucose/metabolism* ; Humans ; Neuroprotective Agents/pharmacology*

This study aims to explore the effects and action mechanisms of the active ingredients in Buyang Huanwu Decoction(BYHWD), namely tetramethylpyrazine(TMP) and hydroxy-safflor yellow A(HSYA), on oxygen-glucose deprivation/reglucose-reoxygenation(OGD/R)-induced inflammation and oxidative stress of microglia(MG). Network pharmacology was used to screen the effective monomer ingredients of BYHWD and determine the safe concentration range for each component. Inflammation and oxidative stress models were established to further screen the best ingredient combination and optimal concentration ratio with the most effective anti-inflammatory and antioxidant effects. OGD/R BV2 cell models were constructed, and BV2 cells in the logarithmic growth phase were divided into a normal group, a model group, an HSYA group, a TMP group, and an HSYA + TMP group. Enzyme-linked immunosorbent assay(ELISA) was used to detect the levels of inflammatory cytokines such as interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6). Oxidative stress markers, including superoxide dismutase(SOD), nitric oxide(NO), and malondialdehyde(MDA), were also measured. Western blot was used to analyze the protein expression of both inflammation-related pathway [Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)] and oxidative stress-related pathway [nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)]. Immunofluorescence was used to assess the expression of proteins such as inducible nitric oxide synthase(iNOS) and arginase-1(Arg-1). The most effective ingredients for anti-inflammatory and antioxidant effects in BYHWD were TMP and HSYA. Compared to the normal group, the model group showed significantly increased levels of IL-1β, TNF-α, IL-6, NO, and MDA, along with significantly higher protein expression of NF-κB, TLR4, Nrf2, and HO-1 and significantly lower SOD levels. The differences between the two groups were statistically significant. Compared to the model group, both the HSYA group and the TMP group showed significantly reduced levels of IL-1β, TNF-α, IL-6, NO, and MDA, lower expression of NF-κB and TLR4 proteins, higher levels of SOD, and significantly increased protein expression of Nrf2 and HO-1. Additionally, the expression of the M1-type MG marker iNOS was significantly reduced, while the expression of the M2-type MG marker Arg-1 was significantly increased. The results of the HSYA group and the TMP group had statistically significant differences from those of the model group. Compared to the HSYA group and the TMP group, the HSYA + TMP group showed further significant reductions in IL-1β, TNF-α, IL-6, NO, and MDA levels, along with significant reductions in NF-κB and TLR4 protein expression, an increase in SOD levels, and elevated Nrf2 and HO-1 protein expression. Additionally, the expression of the M1-type MG marker iNOS was reduced, while the M2-type MG marker Arg-1 expression increased significantly in the HSYA + TMP group compared to the TMP or HSYA group. The differences in the results were statistically significant between the HSYA + TMP group and the TMP or HSYA group. The findings indicated that the combined use of HSYA and TMP, the active ingredients of BYHWD, can effectively inhibit OGD/R-induced inflammation and oxidative stress of MG, showing superior effects compared to the individual use of either component.
Oxidative Stress/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Animals ; Mice ; Glucose/metabolism* ; Cell Line ; Inflammation/genetics* ; Oxygen/metabolism* ; Pyrazines/pharmacology* ; Microglia/metabolism* ; NF-E2-Related Factor 2/immunology* ; NF-kappa B/immunology* ; Toll-Like Receptor 4/immunology* ; Anti-Inflammatory Agents/pharmacology* ; Humans

BACKGROUND AND OBJECTIVE: Patients with glioma experience a high symptom burden and have diverse palliative care needs. However, the assessment scales used in palliative care remain non-standardized and highly heterogeneous. To evaluate the application patterns of the current scales used in palliative care for glioma, we aim to identify gaps and assess the need for disease-specific scales in glioma palliative care. METHODS: We conducted a systematic search of five databases including PubMed, Web of Science, Medline, EMBASE, and CINAHL for quantitative studies that reported scale-based assessments in glioma palliative care. We extracted data on scale characteristics, domains, frequency, and psychometric properties. Quality assessments were performed using the Cochrane ROB 2.0 and ROBINS-I tools. RESULTS: Of the 3,405 records initially identified, 72 studies were included. These studies contained 75 distinct scales that were used 193 times. Mood (21.7%), quality of life (24.4%), and supportive care needs (5.2%) assessments were the most frequently assessed items, exceeding half of all scale applications. Among the various assessment dimensions, the Distress Thermometer (DT) was the most frequently used tool for assessing mood, while the Short Form-36 Health Survey Questionnaire (SF-36) was the most frequently used tool for assessing quality of life. The Mini Mental Status Examination (MMSE) was the most common tool for cognitive assessment. Performance status (5.2%) and social support (6.8%) were underrepresented. Only three brain tumor-specific scales were identified. Caregiver-focused scales were limited and predominantly burden-oriented. CONCLUSIONS: There are significant heterogeneity, domain imbalances, and validation gaps in the current use of assessment scales for patients with glioma receiving palliative care. The scale selected for use should be comprehensive and user-friendly.
Humans ; Glioma/psychology* ; Palliative Care/methods* ; Quality of Life ; Psychometrics ; Brain Neoplasms/psychology*

Approximately 60% of colorectal cancer (CRC) patients exhibit TP53 mutations, which are strongly associated with tumor progression, chemotherapy resistance, and an unfavorable prognosis. However, targeting p53 has historically been challenging, and currently, there are no approved p53-based therapeutics for clinical use worldwide. In this study, we discovered that ubiquitin carboxyl terminal hydrolase L3 (UCHL3) plays a crucial role in high-level glycolysis, enhanced stem-like properties, and 5-fluorouracil (5-FU) chemoresistance in TP53-mutant CRC by exerting its deubiquitinating enzyme activity to stabilize α-enolase (ENO1) protein. Notably, we identified a newly Food and Drug Administration (FDA)-approved drug, pacritinib, that potently suppresses UCHL3 expression by blocking the janus kinase 2 (JAK2)-signal transducer and activator of transcription 3 (STAT3) pathway in TP53-mutant CRC. Furthermore, Pacritinib was demonstrated to effectively inhibit glycolysis and improve the sensitivity to 5-FU chemotherapy in TP53-mutant CRC. Our findings suggest that targeting the JAK2-STAT3-UCHL3-ENO1 axis is a promising strategy to suppress glycolysis and enhance the efficacy of 5-FU chemotherapy in TP53-mutant CRC. Pacritinib shows potential for clinical application in the treatment of TP53-mutant CRC.

OBJECTIVE: Data on homocysteine (Hcy) status and its determinants are limited among women during pregnancy and postpartum. This cross-sectional study aimed to investigate Hcy levels during pregnancy and postpartum, and to explore the determinants like geographic factor. METHODS: This study was conducted in women at mid-pregnancy, late-pregnancy and postpartum from southern, central and northern China. Approximately 132 women were included in each stratum by the three phases and regions. Plasma Hcy concentrations were assessed using high-performance liquid chromatography (HPLC), with hyperhomocysteinemia defined as > 10.0 µmol/L. Quantile regression was to estimate medians and interquartile ranges ( IQRs), and logistic regression to examine the determinants of hyperhomocysteinemia. RESULTS: For 1,190 women included, the median (IQR) Hcy concentration was 5.66 (4.62, 7.37) μmol/L. The adjusted median in mid-pregnancy, late-pregnancy and postpartum women was 4.75 (4.13, 5.54), 5.72 (4.81, 6.85) and 7.09 (5.65, 8.75) μmol/L, respectively, showing an increasing trend ( P < 0.001). This increasing trend persisted across the three regions. Higher Hcy concentrations were observed in women residing in northern region and those with younger age or lower economic status. A total of 106 (8.9%) women had hyperhomocysteinemia, with a higher prevalence in those residing in northern region (16.0%), or in postpartum women (16.5%). CONCLUSION Hcy levels, varying with geographic region, maternal age and economic status, are increased from mid-pregnancy to late-pregnancy and postpartum, indicating a need to monitor Hcy levels in pregnant and postpartum women to control potential risks related to elevated Hcy levels.
Humans ; Female ; Pregnancy ; Homocysteine/blood* ; China/epidemiology* ; Adult ; Postpartum Period/blood* ; Cross-Sectional Studies ; Hyperhomocysteinemia/blood* ; Young Adult ; Pregnancy Trimester, Third/blood* ; Pregnancy Trimester, Second ; East Asian People

OBJECTIVE To study the intervention effects of Wenshen Tongluo Zhitong Recipe(WTZR)on macrophage senes-cence and senile osteoporosis.METHODS The senescent macrophage model was established using hydrogen peroxide(H2 O2)and subsequently divided into four groups:control,model,low-dose drug-treated serum,and high-dose drug-treated serum.β-galactosidase staining,Western blot and qPCR were employed to evaluate the mRNA expression of senescence markers p21 and p53.ROS staining and JC-1 staining were applied to assess mitochondrial function in macrophages.The mRNA levels of Interleukin(IL)-6,IL-10,CD206,and inducible nitric oxide synthase(iNOS)were determined by qPCR analysis.Immunofluorescence was used to evaluate argi-nase(ARG1)and iNOS protein expressions for assessing the impact of drug-containing serum on macrophage polarization.qPCR analy-sis was conducted to measure osteocalcin(OCN),collagen type Ⅰ alpha 1(Col1a1),runt-related transcription factor 2(Runx2)mRNA levels as osteoblast-related markers;ALP staining along with alizarin red staining were performed to evaluate the effect of macrophage conditioned medium treated with drug-containing serum on bone marrow mesenchymal stromal cell(BMSC)osteogenic differentiation.C57BL/6J mice were randomly allocated into four groups:control group,model group,WTZR low-dose group,and WTZR high-dose group.The senile osteoporosis(SOP)mouse model was established by D-galactose.Micro-CT scanning analyzed fe-mur microstructure while HE staining detected pathological changes in femur bone tissue samples collected from each experimental con-dition.Furthermore,Western blot was used to detect the senescence-related molecules p21 and p53 and the osteogenesis-related mark-ers OCN and Runx2,qPCR analysis measured tibial expression levels of senescence-related molecules(p21,p53)as well as macro-phage polarisation-related molecules(IL-6,iNOS,CD206,and IL-10)to assess the effect of this compound on a mouse model simula-ting SOP.RESULTS Following intervention with serum containing WTZR,there was a significant decrease in the number of senes-cent positive cells compared to the model group.Additionally,there was a notable decrease in p21 and p53 mRNA and proteins expres-sion(P＜0.05,P＜0.01,P＜0.001).Furthermore,drug-containing WTZR effectively inhibited ROS production induced by H2 O2 and mitigated mitochondrial membrane potential reduction in macrophages(P＜0.05,P＜0.001).Treatment with drug-containing WTZR re-sulted in down-regulated mRNA expression of M1-related gene iNOS(P＜0.05)while up-regulating mRNA expression of M2-related genes CD163 and CD206(P＜0.05).The drug-containing WTZR significantly reduced fluorescence intensity for iNOS(P＜0.01)while increasing ARG1(P＜0.05)fluorescence intensity.Moreover,conditioned medium from macrophages treated with drug-containing ser-um increased ALP positive cell count(P＜0.01,P＜0.001),alizarin red positive area(P＜0.05),as well as Col1a1,Runx2 and OCN mRNA expression levels(P＜0.05,P＜0.01).The Tb.N,BMD,and BV/TV were significantly higher in the WTZR group compared to the model group(P＜0.05,P＜0.01,P＜0.001);meanwhile,Tb.Sp was notably lower than that observed in the model group(P＜0.05,P＜0.01);bone trabeculae were significantly improved,increased in number and widened.Additionally,the compound could significantly inhibit the D-galactose induced up-regulation of tibial senescence-related genes and proteins p21 and p53(P＜0.05,P＜0.001,P＜0.0001),promote the expression of osteogenesis-related markers OCN and Runx2 protein(P＜0.01,P＜0.0001),promote the down-regulation of M1 related genes IL-6 and iNOS(P＜0.05),and promote the expression of M2 related genes IL-10 and CD206(P＜0.05,P＜0.01).CONCLUSION Wenshen Tongluo Zhitong Recipe may play an anti-osteoporosis effect by inhibiting macrophage senescence and promoting the osteogenic differentiation of BMSC.

Positive pressure bio-protective equipment is a key protective equipment for high-level biosafety laboratories, which provides comprehensive protection for professionals working in pathogenic microorganisms or other biological contamination environments. This paper summarizes the development status of positive pressure bio-protective equipment at home and abroad, analyzes the existing problems of the current positive pressure bio-protective equipment, and puts forward suggestions, to provide reference for the development and application of positive pressure bio-protective equipment in our country.

Objective:To study the current status of longitudinal extrauterine growth restriction (EUGR) in extremely preterm infants (EPIs) and to develop a prediction model based on clinical data from multiple NICUs.Methods:From January 2017 to December 2018, EPIs admitted to 32 NICUs in North China were retrospectively studied. Their general conditions, nutritional support, complications during hospitalization and weight changes were reviewed. Weight loss between birth and discharge > 1SD was defined as longitudinal EUGR. The EPIs were assigned into longitudinal EUGR group and non-EUGR group and their nutritional support and weight changes were compared. The EPIs were randomly assigned into the training dataset and the validation dataset with a ratio of 7∶3. Univariate Cox regression analysis and multiple regression analysis were used in the training dataset to select the independent predictive factors. The best-fitting Nomogram model predicting longitudinal EUGR was established based on Akaike Information Criterion. The model was evaluated for discrimination efficacy, calibration and clinical decision curve analysis.Results:A total of 436 EPIs were included in this study, with a mean gestational age of (26.9±0.9) weeks and a birth weight of (989±171) g. The incidence of longitudinal EUGR was 82.3%(359/436). Seven variables (birth weight Z-score, weight loss, weight growth velocity, the proportion of breast milk ≥75% within 3 d before discharge, invasive mechanical ventilation ≥7 d, maternal antenatal corticosteroids use and bronchopulmonary dysplasia) were selected to establish the prediction model. The area under the receiver operating characteristic curve of the training dataset and the validation dataset were 0.870 (95% CI 0.820-0.920) and 0.879 (95% CI 0.815-0.942), suggesting good discrimination efficacy. The calibration curve indicated a good fit of the model ( P>0.05). The decision curve analysis showed positive net benefits at all thresholds. Conclusions:Currently, EPIs have a high incidence of longitudinal EUGR. The prediction model is helpful for early identification and intervention for EPIs with higher risks of longitudinal EUGR. It is necessary to expand the sample size and conduct prospective studies to optimize and validate the prediction model in the future.

Objective:To summarize the imaging features of severe unilateral transverse sinus and sigmoid sinus thromboses, and evaluate the efficacy and safety of intravascular interventional therapy in them.Methods:Thirty-seven patients with severe unilateral transverse sinus and sigmoid sinus thromboses clinically mainly manifested as intracranial hypertension and accepted endovascular intervention in Department of Interventional Radiology, First Affiliated Hospital of Zhengzhou University from June 2012 to September 2022 were chosen; their clinical data were retrospectively analyzed and imaging features were summarized. Short-term efficacy was evaluated according to blood flow restoration degrees and pressure gradient reduction in the occlusive sinus and modified neurological symptoms before and after endovascular intervention. Hospitalized complications were observed; safety and long-term efficacy were evaluated according to postoperative clinical follow-up and imaging results 6-12 months after endovascular intervention.Results:(1) Preoperative brain MRI and (or) CT showed different degrees of swelling of the brain tissues, with the affected side as the target; mixed signals/density shadow could be seen in the blocked transverse sinus and sigmoid sinus; venous cerebral infarction or post-infarction cerebral hemorrhage could be combined in some patients. MRV, CTV and DSA showed poor or completely occluded transverse sinus and sigmoid sinus while normal in the contralateral side; obvious thrombus filling-defect was observed in the occluded venous sinus after mechanical thrombolysis. (2) Occlusive sinus blood flow was restored in all patients after endovascular intervention, and pressure gradient of the occlusive segment decreased from (16.6±3.3) mmHg before to (2.8±0.8) mmHg after endovascular intervention. Before discharge, clinical symptoms of all patients were significantly improved (modified Rankin scale [mRS] scores of 0 in 30 patients, 1 in 5 patients, 2 in 1 patient and 3 in 1 patient), and 2 patients had unilateral limb movement disorder (muscle strength grading III and IV, respectively). All patients received clinical follow-up for (9.6±3.0) months. At the last follow-up, neurological function obviously improved compared with that before endovascular intervention, without new neurosystem-related symptoms (mRS scores of 0 in 30 patients, 1 in 6, and 2 in 1 patient). In 34 patients received MRV or DSA follow-up, 28 had complete recanalization of occlusive sinus and 6 had partial recanalization, without obvious stenosis or recurrent occlusion.Conclusions:Severe unilateral transverse sinus and sigmoid sinus thrombosis can cause local intracranial venous blood stasis, and then cause "increased regional venous sinus pressure", which is manifested as unilateral brain tissue swelling and even venous cerebral infarction or post-infarction cerebral hemorrhage. Early diagnosis and endovascular intervention can obviously improve the prognosis of these patients, enjoying good safety.