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

Constructing an efficient and easy-to-operate multigene expression system is currently a crucial part of plant genetic engineering. In this study, a fragment carrying three independent gene expression cassettes and the expression unit of the gene-silencing suppressor protein(RNA silencing suppressor 19 kDa protein, P19) simultaneously was designed and constructed. This fragment was cloned into the commonly used plant expression vector pCAMBIA300, and the plasmid pC1300-TP2-P19 was obtained. Each gene expression cassette consists of different promoters, fusion tags, and terminators. The target gene can be flexibly inserted into the corresponding site through enzymatic digestion and ligation or recombination and fused with different protein tags, which provides great convenience for subsequent detection. The enhanced green fluorescent protein(eGFP) reporter gene was individually constructed into each expression cassette to verify the feasibility of this vector system. The results of tobacco transient expression and laser-confocal microscopy showed that each expression cassette presented independent and normal expression. Meanwhile, the three key enzyme genes in the betanin synthesis pathway, BvCYP76AD, BvDODA1, and DbDOPA5GT, were constructed into the three expression cassettes. The results of tobacco transient expression phenotype, protein immunoblotting(Western blot), and chemical detection of product demonstrated that the three exogenous genes were highly expressed, and the target compound betanin was successfully produced. The above results indicated that the constructed multigene expression system for plants in this study was efficient and reliable and can achieve the co-transformation of multiple plant genes. It can provide a reliable vector platform for the analysis of plant natural product synthesis pathways, functional verification, and plant metabolic engineering.
Nicotiana/metabolism* ; Genetic Vectors/metabolism* ; Gene Expression Regulation, Plant ; Plant Proteins/metabolism* ; Plants, Genetically Modified/metabolism* ; Genetic Engineering/methods* ; Green Fluorescent Proteins/metabolism* ; Gene Expression

Schizophrenia (SZ) stands as a severe psychiatric disorder. This study applied diffusion tensor imaging (DTI) data in conjunction with graph neural networks to distinguish SZ patients from normal controls (NCs) and showcases the superior performance of a graph neural network integrating combined fractional anisotropy and fiber number brain network features, achieving an accuracy of 73.79% in distinguishing SZ patients from NCs. Beyond mere discrimination, our study delved deeper into the advantages of utilizing white matter brain network features for identifying SZ patients through interpretable model analysis and gene expression analysis. These analyses uncovered intricate interrelationships between brain imaging markers and genetic biomarkers, providing novel insights into the neuropathological basis of SZ. In summary, our findings underscore the potential of graph neural networks applied to multimodal DTI data for enhancing SZ detection through an integrated analysis of neuroimaging and genetic features.
Humans ; Schizophrenia/pathology* ; Diffusion Tensor Imaging/methods* ; Male ; Female ; Adult ; Brain/metabolism* ; Young Adult ; Middle Aged ; White Matter/pathology* ; Gene Expression ; Nerve Net/diagnostic imaging* ; Graph Neural Networks

Objective To investigate the clinical characteristics and prognosis of pneumococcal meningitis(PM),and drug sensitivity of Streptococcus pneumoniae(SP)isolates in Chinese children.Methods A retrospective analysis was conducted on clinical information,laboratory data,and microbiological data of 160 hospitalized children under 15 years old with PM from January 2019 to December 2020 in 33 tertiary hospitals across the country.Results Among the 160 children with PM,there were 103 males and 57 females.The age ranged from 15 days to 15 years,with 109 cases(68.1% )aged 3 months to under 3 years.SP strains were isolated from 95 cases(59.4% )in cerebrospinal fluid cultures and from 57 cases(35.6% )in blood cultures.The positive rates of SP detection by cerebrospinal fluid metagenomic next-generation sequencing and cerebrospinal fluid SP antigen testing were 40% (35/87)and 27% (21/78),respectively.Fifty-five cases(34.4% )had one or more risk factors for purulent meningitis,113 cases(70.6% )had one or more extra-cranial infectious foci,and 18 cases(11.3% )had underlying diseases.The most common clinical symptoms were fever(147 cases,91.9% ),followed by lethargy(98 cases,61.3% )and vomiting(61 cases,38.1% ).Sixty-nine cases(43.1% )experienced intracranial complications during hospitalization,with subdural effusion and/or empyema being the most common complication[43 cases(26.9% )],followed by hydrocephalus in 24 cases(15.0% ),brain abscess in 23 cases(14.4% ),and cerebral hemorrhage in 8 cases(5.0% ).Subdural effusion and/or empyema and hydrocephalus mainly occurred in children under 1 year old,with rates of 91% (39/43)and 83% (20/24),respectively.SP strains exhibited complete sensitivity to vancomycin(100% ,75/75),linezolid(100% ,56/56),and meropenem(100% ,6/6).High sensitivity rates were also observed for levofloxacin(81% ,22/27),moxifloxacin(82% ,14/17),rifampicin(96% ,25/26),and chloramphenicol(91% ,21/23).However,low sensitivity rates were found for penicillin(16% ,11/68)and clindamycin(6% ,1/17),and SP strains were completely resistant to erythromycin(100% ,31/31).The rates of discharge with cure and improvement were 22.5% (36/160)and 66.2% (106/160),respectively,while 18 cases(11.3% )had adverse outcomes.Conclusions Pediatric PM is more common in children aged 3 months to under 3 years.Intracranial complications are more frequently observed in children under 1 year old.Fever is the most common clinical manifestation of PM,and subdural effusion/emphysema and hydrocephalus are the most frequent complications.Non-culture detection methods for cerebrospinal fluid can improve pathogen detection rates.Adverse outcomes can be noted in more than 10% of PM cases.SP strains are high sensitivity to vancomycin,linezolid,meropenem,levofloxacin,moxifloxacin,rifampicin,and chloramphenicol.[Chinese Journal of Contemporary Pediatrics,2024,26(2):131-138]

Objective To investigate the incidence rate,clinical characteristics,and prognosis of neonatal stroke in Shenzhen,China.Methods Led by Shenzhen Children's Hospital,the Shenzhen Neonatal Data Collaboration Network organized 21 institutions to collect 36 cases of neonatal stroke from January 2020 to December 2022.The incidence,clinical characteristics,treatment,and prognosis of neonatal stroke in Shenzhen were analyzed.Results The incidence rate of neonatal stroke in 21 hospitals from 2020 to 2022 was 1/15 137,1/6 060,and 1/7 704,respectively.Ischemic stroke accounted for 75％(27/36);boys accounted for 64％(23/36).Among the 36 neonates,31(86％)had disease onset within 3 days after birth,and 19(53％)had convulsion as the initial presentation.Cerebral MRI showed that 22 neonates(61％)had left cerebral infarction and 13(36％)had basal ganglia infarction.Magnetic resonance angiography was performed for 12 neonates,among whom 9(75％)had involvement of the middle cerebral artery.Electroencephalography was performed for 29 neonates,with sharp waves in 21 neonates(72％)and seizures in 10 neonates(34％).Symptomatic/supportive treatment varied across different hospitals.Neonatal Behavioral Neurological Assessment was performed for 12 neonates(33％,12/36),with a mean score of(32±4)points.The prognosis of 27 neonates was followed up to around 12 months of age,with 44％(12/27)of the neonates having a good prognosis.Conclusions Ischemic stroke is the main type of neonatal stroke,often with convulsions as the initial presentation,involvement of the middle cerebral artery,sharp waves on electroencephalography,and a relatively low neurodevelopment score.Symptomatic/supportive treatment is the main treatment method,and some neonates tend to have a poor prognosis.

Objective:To explore diagnostic value of echocardiography indexes for moderate-severe stenosis in CHD patients and their correlation with cardiac function class.Methods:A total of 90 CHD patients admitted to our hos-pital from Dec 2020 to Dec 2022 were selected as CHD group.According to NYHA classification,CHD group was divided into class Ⅰ～Ⅱ group(n=38)and class Ⅲ～Ⅳ group(n=52).The patients were divided into mild group(n=35)and moderate-severe group(n=55)according to Gensini score.In addition,90 healthy people who sim-ultaneously received physical examination in our hospital were enrolled as control group.LVEF,LVEDd and LVESd were measured in two groups by color ultrasound diagnostic instrument.Cardiac function indexes were com-pared between control group and CHD group,mild group and moderate-severe group,class Ⅰ～Ⅱ group and classⅢ～Ⅳ group.ROC curve was used to analyze the diagnostic value of above cardiac function indexes for moderate-severe stenosis in CHD patients.The correlation among LVEF,LVEDd,LVESd,different stenosis degree and NYHA class in CHD patients was analyzed by Spearman correlation analysis.Results:Compared with the control group,there was significant reduction in LVEF,and significant rise in LVEDd and LVESd in CHD group,P=0.001 all;compared with the mild group,there was significant reduction in LVEF[(45.31±5.08)％vs.(40.34±3.01)％],and significant rise in LVEDd[(53.92±5.09)mm vs.(61.68±4.79)mm]and LVESd[(52.72±4.72)mm vs.(58.06±3.50)mm]in moderate-severe group,P=0.001 all;compared with the class Ⅰ～Ⅱgroup,there was significant reduction in LVEF[(45.07±4.95)％vs.(40.23±3.06)％],and significant rise in LVEDd[(54.50±5.30)mm vs.(61.71±4.91)mm]and LVESd[(52.92±4.63)mm vs.(58.22±3.43)mm]in class Ⅲ～Ⅳ group,P=0.001 all.AUC of combined detection of LVEF,LVEDd and LVESd diagnosing moder-ate-severe stenosis in CHD was 0.909,which was significantly higher than those of single detections(0.733,0.787,0.789)(Z=2.925,2.125,2.043,P＜0.05 or＜0.01).Spearman correlation analysis showed a significant negative correlation between LVEF and NYHA class(r=-0.514),LVEDd and LVESd showed a significant posi-tive correlation with NYHA class(r=0.538,0.546,P=0.001 both),and it showed a significant positive correla-tion between different degrees of coronary stenosis and NYHA class in CHD patients(r=0.875,P=0.001).Con-clusion:The combined detection of LVEF,LVEDd and LVESd possesses high diagnostic value for moderate-se-vere stenosis in CHD patients,and it is significantly correlated with NYHA cardiac function class,which can be used as one of assessing methods for coronary stenosis severity in CHD patients.

ObjectiveOur recent study has demonstrated that extracellular acidification promotes lipid accumulation in macrophages via the activation of acid sensing ion channel 1 (ASIC1), but the underlying mechanism remains unclear. This study aims to explore the effect of extracellular acidification on macrophage lipophagy and the underlying mechanism. MethodsRAW264.7 macrophages were incubated with 25 mg/Lox-LDL in a pH 6.5 culture medium for 24 h to build macrophage-derived foam cell models induced by extracellular acidification. Then, RAW264.7 macrophages were cultured in the acidic medium of pH 6.5 with or without PcTx-1 (ASIC1 specific blocker, 10 μg/L) or Nec-1 (RIP1 specific inhibitor, 20 μmol/L) for 24 h, intracellular lipid accumulation was observed by oil red O staining. The expressions of total ASIC1, plasma membrane ASIC1, RIP1, p-RIP1 Ser166, TFEB, p-TFEB Ser142, LC3 and p62 were measured by Western blot. The co-localization of lipids (indicated by Bodipy) with LC3II (autophagosomes) and LAMP1 (lysosomes) was analyzed by a confocal laser scanning microscopy, respectively. Morphological changes of lipophagy in the cells were observed by using transmission electron microscopy. ABCA1-mediated cholesterol efflux was determined by cholesterol fluorescence kits. ResultsCompared with pH 7.4+ox-LDL group, the intracellular lipid accumulation in the pH 6.5+ox-LDL group was significantly increased. Meanwhile, the expressions of plasma membrane ASIC1, p-RIP1 Ser166, p-TFEB Ser142, and p62 proteins were elevated significantly, while LC3II protein level and LC3II/LC3I ratio were decreased. Accordingly, compared with pH 7.4+ox-LDL group, the macrophage lipophagy of the pH 6.5+ox-LDL group was inhibited as indicated by the decreased localization of lipid droplets with LC3 and LAMP1, a decrease in the number of lipophagosomes as well as an increase in lipid droplets. Furthermore, ATP binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux from the macrophages of pH 6.5+ox-LDL group reduced dramatically. However, these above effects of extracellular acidification on RAW264.7 macrophages were abolished by PcTx-1 and Nec-1, respectively. ConclusionThese findings suggest extracellular acidification promotes the phosphorylation of TFEB at Ser142 via activating ASIC1/RIP1 pathway, thereby impeding lipophagy in RAW 264.7 macrophages, and that ASIC1 may be a new potential target for preventing aberrant lipid accumulation diseases including atherosclerosis.

Objective To analyze the TCM syndrome and treatment rule for the treatment of chronic atrophic gastritis based on data mining;To provide reference for its clinical treatment.Methods With the help of the big data platform of evidence-based literature of TCM covering several dominant diseases in the field of digestion,literature on the experience of famous doctors,theoretical discussion and case reports in the treatment of chronic atrophic gastritis published from 1 January 2000 to 31 July 2022 was retrieved.Medical records were screened according to the inclusion and exclusion criteria.The data of TCM disease names,clinical symptoms,tongue manifestation,pulse manifestation,TCM syndrome,TCM treatment method and other terms in medical records were standardized and statistically analyzed.Results Totally 169 articles were included,including 228 medical cases,involving 228 patients,with a cumulative number of 361 visits,with an average age of(53.17±11.11)years old,with the largest number of 50-60 years old.Chronic atrophic gastritis mostly belonged to the categories of"stomach pain"and"epigastric puffiness"in TCM,and its main symptoms were stomach pain,epigastric puffiness and belching.The common tongue manifestation include dark red tongue,white moss,thin or greasy moss.The common pulse manifestation included wiry slippery pulse,wiry thready pulse and wiry pulse.The high-frequency syndromes included liver-stomach qi stagnation syndrome,spleen-stomach dampness-heat syndrome,liver-qi stagnation and spleen-qi deficiency syndrome.The disease locations were mainly in the stomach,spleen and liver,and the syndrome elements were mainly qi deficiency,qi stagnation,blood stasis,dampness and heat,etc.The complex syndrome elements were more than the single syndrome elements,and the two syndrome elements of qi deficiency + qi stagnation was the most common.The most commonly used treatment methods were supplementing,regulating qi and regulating blood.Conclusion The treatment of chronic atrophic gastritis with TCM mostly focuses on the stomach,spleen and liver.The core syndrome elements are qi deficiency,qi stagnation,and blood stasis.The methods of nourishing qi and strengthening the spleen,soothing the liver and stomach,regulating qi and blood are often used.

Objective To compare the effects of 20 mg qd and 10 mg bidadministration of iprrazole enteric-coated tablets on the control of gastric acid in healthy subjects.Methods A randomized,single-center,parallel controlled trial was designed to include 8 healthy subjects.Randomly divided into 2 groups,20 mg qd administration group:20 mg enteric-coated tablets of iprrazole in the morning;10 mg bid administration group:10 mg enteric-coated tablets of iprrazole in the morning and 10 mg in the evening.The pH values in the stomach of the subjects before and 24 h after administration were monitored by pH meter.The plasma concentration of iprazole after administration was determined by HPLC-MS/MS.The main pharmacokinetic parameters were calculated by Phoenix WinNonlin(V8.0)software.Results The PK parameters of iprrazole enteric-coated tablets and reference preparations in fasting group were as follows:The Cmax of 20 mg qd group and 10 mg bid group were(595.75±131.15)and(283.50±96.98)ng·mL-1;AUC0-t were(5 531.94±784.35)and(4 686.67±898.23)h·ng·mL-1;AUC0-∞ were(6 003.19±538.59)and(7 361.48±1 816.77)h·ng·mL-1,respectively.The mean time percentage of gastric pH＞3 after 20 mg qd and 10 mg bid were 82.64％and 61.92％,and the median gastric pH within 24 h were 6.25±1.49 and 3.53±2.05,respectively.The mean gastric pH values within 24 h were 5.71±1.36 and 4.23±1.45,respectively.The correlation analysis of pharmacokinetic/pharmacodynamics showed that there was no significant correlation between the peak concentration of drug in plasma and the inhibitory effect of acid.Conclusion Compared with the 20 mg qd group and the 10 mg bid group,the acid inhibition effect is better,the administration times are less,and the safety of the two administration regimes is good.

Objective To evaluate the efficacy and safety of brexpiprazole in treating acute schizophrenia.Methods Patients with schizophrenia were randomly divided into treatment group and control group.The treatment group was given brexpiprozole 2-4 mg·d-1 orally and the control group was given aripiprazole 10-20 mg·d-1orally,both were treated for 6 weeks.Clinical efficacy of the two groups,the response rate at endpoint,the changes from baseline to endpoint of Positive and Negative Syndrome Scale(PANSS),Clinical Global Impression-Improvement(CGI-S),Personal and Social Performance scale(PSP),PANSS Positive syndrome subscale,PANSS negative syndrome subscale were compared.The incidence of treatment-related adverse events in two groups were compared.Results There were 184 patients in treatment group and 186 patients in control group.After treatment,the response rates of treatment group and control group were 79.50％(140 cases/184 cases)and 82.40％(150 cases/186 cases),the scores of CGI-I of treatment group and control group were(2.00±1.20)and(1.90±1.01),with no significant difference(all P＞0.05).From baseline to Week 6,the mean change of PANSS total score wese(-30.70±16.96)points in treatment group and(-32.20±17.00)points in control group,with no significant difference(P＞0.05).The changes of CGI-S scores in treatment group and control group were(-2.00±1.27)and(-1.90±1.22)points,PSP scores were(18.80±14.77)and(19.20±14.55)points,PANSS positive syndrome scores were(-10.30±5.93)and(-10.80±5.81)points,PANSS negative syndrome scores were(-6.80±5.98)and(-7.30±5.15)points,with no significant difference(P＞0.05).There was no significant difference in the incidence of treatment-related adverse events between the two group(69.00％vs.64.50％,P＞0.05).Conclusion The non-inferiority of Brexpiprazole to aripiprazole was established,with comparable efficacy and acceptability.