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Tanshinones are one of the main effective components of Salvia miltiorrhiza, which play important roles in the treatment of cardiovascular diseases. Microbial heterogony production of tanshinones can provide a large number of raw materials for the production of traditional Chinese medicine(TCM) preparations containing S. miltiorrhiza, reduce the extraction cost, and relieve the pressure of clinical medication. The biosynthetic pathway of tanshinones contains multiple P450 enzymes, and the catalytic element with high efficiency is the basis of microbial production of tanshinones. In this study, the protein modification of CYP76AK1, a key P450-C20 hydroxylase in tanshinone pathway, was researched. The protein modeling methods SWISS-MODEL, Robetta, and AlphaFold2 were used, and the protein model was analyzed to obtain the reliable protein structure. The semi-rational design of mutant protein was carried out by molecular docking and homologous alignment. The key amino acid sites affecting the oxidation activity of CYP76AK1 were identified by molecular docking. The function of the obtained mutations was studied with yeast expression system, and the CYP76AK1 mutations with continuous oxidation function to 11-hydroxysugiol were obtained. Four key amino acid sites that affected the oxidation acti-vity were analyzed, and the reliability of three protein modeling methods was analyzed according to the mutation results. The effective protein modification sites of CYP76AK1 were reported for the first time in this study, which provides a catalytic element for different oxidation activities at C20 site for the study of the synthetic biology of tanshinones and lays a foundation for the analysis of the conti-nuous oxidation mechanism of P450-C20 modification.
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Oxidoreductases , Biosynthetic Pathways , Molecular Docking Simulation , Reproducibility of Results , Salvia miltiorrhiza/chemistry , Amino Acids/metabolism , Plant Roots/geneticsABSTRACT
Retinopathy of prematurity (ROP) is a blinding eye disease characterized by abnormal proliferation of retinal blood vessels in children.Vascular endothelial growth factor (VEGF) can specifically stimulate retinal vascular endothelial cell proliferation and neovascularization.Retinal ischemia and hypoxia in preterm infants promote the compensatory increase of intraocular VEGF expression, and then induce the pathological growth of retinal vessels.The intravitreal injection of anti-VEGF drugs provides a new therapeutic way for ROP by inhibiting the biological activity of VEGF and delaying retinal neovascularization.However, the blood-retina barrier of children with ROP is likely to be destructed, which can cause the imbalanced homeostasis of the retinal microenvironment.The anti-VEGF drugs can cause irreversible damage to nerve cells through the blood-retina barrier and blood-brain barrier, which affects the development of the nervous system in children with ROP.At present, whether anti-VEGF drugs result in the abnormal development and functional changes of nervous system in premature infants remains unknown and attracts much attention.In this paper, the role of VEGF in the pathogenesis of ROP and neurodevelopment, as well as the effects of anti-VEGF drug intravitreal injections on the neurological development of children with ROP were reviewed to provide the clinical basis for the rational and safe application of anti-VEGF drugs.
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Ferroptosis is a form of regulated cell death, characterized by excessive membrane lipid peroxidation in an iron- and ROS-dependent manner. Celastrol, a natural bioactive triterpenoid extracted from Tripterygium wilfordii, shows effective anti-fibrotic and anti-inflammatory activities in multiple hepatic diseases. However, the exact molecular mechanisms of action and the direct protein targets of celastrol in the treatment of liver fibrosis remain largely elusive. Here, we discover that celastrol exerts anti-fibrotic effects via promoting the production of reactive oxygen species (ROS) and inducing ferroptosis in activated hepatic stellate cells (HSCs). By using activity-based protein profiling (ABPP) in combination with bio-orthogonal click chemistry reaction and cellular thermal shift assay (CETSA), we show that celastrol directly binds to peroxiredoxins (PRDXs), including PRDX1, PRDX2, PRDX4 and PRDX6, through the active cysteine sites, and inhibits their anti-oxidant activities. Celastrol also targets to heme oxygenase 1 (HO-1) and upregulates its expression in activated-HSCs. Knockdown of PRDX1, PRDX2, PRDX4, PRDX6 or HO-1 in HSCs, to varying extent, elevated cellular ROS levels and induced ferroptosis. Taken together, our findings reveal the direct protein targets and molecular mechanisms via which celastrol ameliorates hepatic fibrosis, thus supporting the further development of celastrol as a promising therapeutic agent for liver fibrosis.
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Objective:To compare the thickness of the macular ganglion cell inner plexiform layer (mGCIPL) in patients with a history of laser photocoagulation (LP) versus intravitreal injection of ranibizumab (IVR) for retinopathy of prematurity (ROP).Methods:A retrospective clinical study. From June 2020 to January 2021, 70 eyes of 35 children with a history of surgery for ROP in Shenzhen Eye Hospital were included in the study. Among them, 18 males had 36 eyes, and 17 females had 34 eyes. The average age was 5.54±1.04 years. There were 18 patients (36 eyes) in LP group and 17 patients (34 eyes) in IVR group. There was no significant difference in age ( t=-1.956), sexual composition ratio ( χ2=0.030), birth gestational age ( t=-1.316) and birth weight ( t=-1.060) between the two groups ( P=0.059, 0.862, 0.197, 0.297). All the eyes underwent the examination of optical coherence tomography (OCT). An elliptical region of 14.13 mm 2 centered on macular fovea was scanned according to the macular cube 512×128 model of the Cirrus HD-OCT 5000. The software was used to automatically divide macular fovea into six sectors (superior, inferior, temporal-superior, temporal-inferior, nasal-superior and nasal-inferior) and the average and minimum thickness of mGCIPL. t test was used to compared mGCIPL thickness between two groups using independent samples. Pearson correlation analysis was used to evaluate the correlation between mGCIPL thickness and age, birth gestational age, birth weight. Results:Patients in IVR group had significantly decreased mGCIPL thickness than that in LP group in the six sectors (superior, inferior, temporal-superior, temporal-inferior, nasal-superior and nasal-inferior) and the average and minimum ( t=6.484, 6.719, 7.682, 7.697, 5.151, 5.008, 7.148, 6.581; P<0.05). The thickness of mGCIPL was not significantly correlated with age, birth gestational age, birth weight ( P>0.05). Conclusion:The thickness of mGCIPL in patients with IVR treatment history is thinner than that in LP treatment.
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Retinopathy of prematurity (ROP) is one of the leading causes of visual impairment in children. As understanding on the pathogenesis of ROP accumulated, anti-vascular endothelial growth factor (VEGF) drugs and their application have changed the treatment mode. Anti-VEGF therapy, with convenient operation and clear efficacy, has become an important treatment method for ROP. However, due to the dysfunction of organs in children with ROP, anti-VEGF drugs can enter blood circulation after intravitreal injection and then lead to temporarily reduction of the VEGF level in the blood, which may theoretically cause adverse effects on the development of all organs (especially the brain) in children with ROP. Therefore, it's necessary to pay attention to the effect of anti-VEGF drugs on neurodevelopment in children with ROP, strictly grasp the indications, and standardize its clinical application, so as to continuously improve the overall prognosis of ROP.
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Camptotheca acuminata produces camptothecin (CPT), a monoterpene indole alkaloid (MIA) that is widely used in the treatment of lung, colorectal, cervical, and ovarian cancers. Its biosynthesis pathway has attracted significant attention, but the regulation of CPT biosynthesis by the APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors (TFs) remains unclear. In this study, a systematic analysis of the AP2/ERF TFs family in C. acuminata was performed, including phylogeny, gene structure, conserved motifs, and gene expression profiles in different tissues and organs (immature bark, cotyledons, young flower, immature fruit, mature fruit, mature leaf, roots, upper stem, and lower stem) of C. acuminata. A total of 198 AP2/ERF genes were identified and divided into five relatively conserved subfamilies, including AP2 (26 genes), DREB (61 genes), ERF (92 genes), RAV (18 genes), and Soloist (one gene). The combination of gene expression patterns in different C. acuminata tissues and organs, the phylogenetic tree, the co-expression analysis with biosynthetic genes, and the analysis of promoter sequences of key enzymes genes involved in CPT biosynthesis pathways revealed that eight AP2/ERF TFs in C. acuminata might be involved in CPT synthesis regulation, which exhibit relatively high expression levels in the upper stem or immature bark. Among these, four genes (CacAP2/ERF123, CacAP2/ERF125, CacAP2/ERF126, and CacAP2/ERF127) belong to the ERF-B2 subgroup; two genes (CacAP2/ERF149 and CacAP2/ERF152) belong to the ERF-B3 subgroup; and two more genes (CacAP2/ERF095 and CacAP2/ERF096) belong to the DREB-A6 subgroup. These results provide a foundation for future functional characterization of the AP2/ERF genes to enhance the biosynthesis of CPT compounds of C. acuminata.
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Objective: To optimize the extraction technology and inclusion process of volatile oil for Qingxiangrukang Granules (QG). Methods: Box-Behnken was used to optimize the extraction technology of volatile oil in QG. With yield of inclusion compound and inclusion rate of volatile oil-β-CD as evaluation indexes, and with volatile oil-pure water ratio, β-CD-volatile oil ratio, and inclusion time as investigate factors, the optimal inclusion technology for volatile oil of QG was ensured based on PCA-G1-entropy method and orthogonal design and by using colloid milling. Results: The optimum extraction technology of volatile oil were as follows: extracting time was 6 h, liquid-material ratio was 10, immersion was 0 h. The optimal inclusion technology for volatile oil were as follows: pure water-volatile oil ratio was 1:80, β-CD-volatile oil ratio was 6 g, and the inclusion time was 30 min. Under such condition, there was no significant difference between verification groups of three batches. Conclusion: This optimal extraction technology and inclusion process is stable and workable and can provide experimental basis for industrial production of QG.
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Objective To study the detection rate of pathogens from sputum , blood, and bronchoalveolar lavage fluid ( BALF ) samples in acquired immunodeficiency syndrome ( AIDS ) patients complicated with pulmonary infection.Methods Seventy-three hospitalized AIDS patients complicated with pulmonary infection in Beijing Ditan Hospital , Capital Medical University were enrolled from February 2018 to September 2018.Blood, sputum and BALF samples were collected.Blood samples were cultured to detect anaerobic bacteria, aerobic bacteria, fungi and mycobacteria.Antigen agglutination method was applied in blood samples to detect cryptococcus neoformans.The sputum samples were tested for Mycobacterium tuberculosis by acid-fast staining and were cultured to detect bacteria and fungi.The sputum samples were observed under microscope for sporotrichosis and fungal spores.The BALF samples were cultured to detect bacteria and fungi. The BALF samples were tested for Mycobacterium tuberculosis by polymerase chain reaction amplification and acid-fast staining.Pneumocystis were detected in BALF samples by methenamine silver staining method .The BALF samples were observed under a microscope for sporotrichosis and fungal spores .The detection rate of pathogens from blood, sputum and BALF samples were compared.Chi-square test was conducted for statistical analysis.Results In 73 AIDS patients complicated with pulmonary infection , the pathogen detection rates in blood, sputum and BALF samples were 8 (11.0%), 23 ( 31.5%) and 48 (65.8%), respectively.The difference was statistically significant ( F ＝48.513, P ＜0.01 ).The detection rate in BALF samples was significantly higher than that in blood or sputum samples ( χ2 ＝43.349 and 17.136, respectively, both P＜0.01).The detection rate in sputum samples was significantly higher than that in blood (χ2 ＝9.215, P＜0.05). The highest detection rates of pathogens in blood , sputum and BALF samples were Talaromyces marneffei 4.1%(3), viridans group streptococci 16.4%(12) and 35.6%(26), respectively.Conclusions The detection rate of pathogens in BALF samples from AIDS patients complicated with pulmonary infection is the highest , followed by sputum and blood samples.
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OBJECTIVE Glioblastoma multiforme (GBM) is the most malignant primary tumor of the central nervous system and is associated with a very poor prognosis. No further improvements in outcomes have been reported since radiotherapy-temozolomide therapy was introduced.Therefore,de-veloping new agents to treat GBM is important. This study aimed to evaluate the anti-tumor effect of evodiamine (Evo) on GBM cells, and to determine the underlying mechanisms involved. METHODS U251,LN229,HEB and PC12 cells were treated with various concentrations of evodiamine for 24 and 48 hours,cell viability was measured by MTT assay.The U251 and LN229 cells were treated with evo-diamine(0-10 μmol·L-1)for 24 h,and then stained with Hoechst 33258.An Annexin V-FITC Apoptosis Detection Kit was used to detect apoptosis in the cells.Reactive oxygen species(ROS)production was detected using dichlorofluorescein diacetate (DCFH-DA) staining. The changes in mitochondrial mem-brane potential (MMP) were assessed by JC-1 after cells were treated with evodiamine. The expres-sion levels of p-PI3K,PI3K,p-Akt,Akt,Bax,Bcl-2,p-p38,p38,p-JNK,JNK,p-ERK,ERK,Cytochrome c, Caspase-3, cleaved Caspase-3, PRAP, and cleaved PARP were measured by Western blot analy-ses. RESULTS According to MTT assay results, Evo significantly inhibited the cell proliferation in a time- and dose-dependent manner. Fluorescence microscopy and flow cytometry analyses revealed that Evo induced cell apoptosis in a concentration-dependent manner.Moreover,Evo induced reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) disruption. Finally, Evo induced apoptosis in cancer cells by suppressing PI3K/AKT signaling and inducing MAPK phos-phorylation(p38 and JNK,but not ERK)to regulate apoptotic proteins(Bax,Bcl-2,Cytochrome c,Cas-pase-3, and PARP). CONCLUSION In summary, Evo inhibits cell proliferation by inducing cellular apoptosis via suppressing PI3K/AKT and activating MAPK in GBM;these results indicate that Evo may be regarded as a new approach for GBM treatment.
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Objective To investigate the effect of compound polyethylene glycol electrolyte powder (SF-PEG) and Magnesium Sulfate (MgSO4) on intestinal tract preparation in patients with constipation. Methods 135 cases of constipation patients who underwent colonoscopy were selected. They were divided into A, B and C groups, each with 45 cases.Group A and group B received oral SF-PEG and 219.2 g (2 000 ml) at 5:00 to 7:00. Group A take 50% MgSO450 ml at 9:00, and then took 250 ml warm water.Group B received the same dose of MgSO4 at 7:00. The two groups were examined by colonoscopy at 11:00. Group C received oral compound SF-PEG 219.2 g (2 000 ml) at 10:00 to 12:00, and then took 50% MgSO450 ml at 14:00, received colonoscopy at 16:00. According to the Boston bowel preparation scale (BBPS) score and parallel intraluminal bubble score. Determine the duration of bowel preparation process,and tolerance and adverse effects were recorded during bowel preparation. Results All patients completed bowel preparation and underwent a colonoscopy successfully. The scores of group A in BBPS were significantly higher than those in group B and group C (P < 0.05). In group A and group B, the score of parallel intraluminal bubble was lower than C, which was statistically significant (P < 0.05). The intestinal preparation time of A and B was less than that of group C, which was statistically significant (P < 0.05), and the tolerance of patients was higher in group A and group B than that in group C (P < 0.05). In adverse reactions, group A and group B were lower than those in group C (P < 0.05). Conclusions When taking the time (5:00 to 7:00), intermittent polyethylene glycol electrolyte powder and Magnesium Sulfate can shorten the bowel preparation time and improve the quality of bowel preparation in patients with constipation.
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The purpose of this article is to provide an overview of precision nursing and its implications for education,practice and research.The emphases are on the contributions of nursing practice and research to genomics as well as precision health and precision medicine.Recommendations are elucidated on how to build knowledge and competence of precision nursing within the broad arena of precision health and precision medicine to promote the development of precision nursing.
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<p><b>OBJECTIVE</b>To observe the seasonal changes in serum levels of interleukin-1 beta (IL-1β), interleukin-6 (IL-6) and melatonin (MT) in Bizheng rat model, and explore the relationship between MT and the pathogenesis of rheumatoid arthritis.</p><p><b>METHODS</b>One hundred and sixty Sprague-Dawley rats were randomly divided into four groups in summer (n=80) and winter (n=80) respectively: normal group, collagen-induced arthritis (CIA) model group, operation group, and sham-operation group (n=20 in each group). The CIA model group was injected with collagen emulsion at the base of the tail to induce arthritis. The rats in the operation group received pineal gland resection, and 7 days after the first operation, underwent testectomy or oophorectomy. The rats in the sham-operation group were operated to ligature the sagittal sinus, without extracting the pineal gland. After the operations, the operation group and the sham-operation group both were immunized as the CIA group was. The serum levels of IL-1β, IL-6 and MT in different groups were measured by radioimmunoassay.</p><p><b>RESULTS</b>Compared with the normal group, the serum levels of IL-1β and IL-6 increased in the CIA model, operation, and sham-operation groups both in summer and in winter (IL-1β in summer, P=0.008, P<0.01, P=0.012; IL-1β in winter, P=0.019, P<0.01, P=0.027; IL-6 in summer, P=0.028, P<0.01, P=0.024; IL-6 in winter, P=0.006, P<0.01, P=0.008). In the operation group, the serum levels of IL-1β and IL-6 in winter were higher than in summer, but with no statistically significant differences (P=0.844, 0.679). Compared with the normal group, the serum level of MT significantly increased in summer and winter in both the CIA model group (P=0.002, 0.008) and the sham-operation group (P=0.003, 0.007), while significantly decreased in the operation group (P=0.023, 0.003). There was no significant difference in MT level in the operation group between summer and winter (P=0.947).</p><p><b>CONCLUSIONS</b>The increase of serum levels of IL-1β and IL-6 may exacerbate the inflammatory reaction and cause a more severe condition in the rheumatoid arthritis. The concentrations of IL-1β, IL-6, and MT correspond with the change of seasons, confirming that there are connections between nature and human body.</p>