ObjectiveTo investigate the effects of different fermentation methods and times on the fungal flora and chemical composition of Aurantii Fructus， in order to obtain the optimal fermentation conditions and flora structure， and to ensure the stability and controllability of the fermented varieties. MethodsScanning electron microscopy was used to observe and analyze the colony characteristics on the surface of Aurantii Fructus under different fermentation conditions. Internal transcribed spacer 2（ITS2） high-throughput sequencing， combined with fungal community diversity analysis and fungal community structure analysis， were used to obtain the fungal flora microbial categories of Aurantii Fructus under the conditions of traditional pressure-shelf fermentation and non-pressure-shelf natural fermentation for 7， 14， 21 d（numbered Y1-Y3 for the former， and numbered F1-F3 for the latter）， respectively. At the same time， the chemical components in the fermentation process were detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）， combined with principal component analysis（PCA）， partial least squares-discriminant analysis（PLS-DA） and compound retention time， parent ions， characteristic fragment ions and other information， the differential compounds between the different fermentation samples were screened and identified. ResultsThe analysis of fungal community diversity showed that the dominant flora did not change at different fermentation time points in the traditional pressure-shelf fermentation method， while in the non-pressure-shelf natural fermentation method， there was a significant difference with the fermentation process， and at the genus level， the dominant genus of samples Y1， Y2， Y3 and F2 was Aspergillus， while the dominant genera of samples F1 and F3 were both Rhizopus. This indicated that the microbial growth environment provided by the traditional fermentation method was more stable， and the microbial community structure was more stable， which was more conducive to the stable and controllable fermentation process and fermented products. A total of 155 compounds were identified by compositional analysis， including 70 flavonoids， 38 coumarins， 10 alkaloids， 34 organic acids and 3 other compounds. After fermentation， two new components of ribalinine and pranferin were produced. Different fermentation conditions also brought about differences in chemical composition， multivariate statistical analysis obtained 26 differential compounds under two different fermentation methods， mainly including flavonoids， organic acids and coumarins. Comprehensively， the microbial community structure of samples fermented by the traditional pressure-shelf method of Aurantii Fructus for 14 d was stable， the species richness was high and the overall content of differential compounds was high， which was the optimal processing condition. ConclusionCompared with non-pressure-shelf natural fermentation， the traditional method has obvious advantages in terms of the stability of the microbial community structure and the content of chemical compounds， and the optimal condition is 14 days of fermentation. This study is helpful to promote the quality stability and fermentation bioavailability of fermented products of Aurantii Fructus， as well as to provide an experimental basis for the further improvement of the quality control methods of this variety.

ObjectiveTo investigate the effects of different fermentation methods and times on the fungal flora and chemical composition of Aurantii Fructus， in order to obtain the optimal fermentation conditions and flora structure， and to ensure the stability and controllability of the fermented varieties. MethodsScanning electron microscopy was used to observe and analyze the colony characteristics on the surface of Aurantii Fructus under different fermentation conditions. Internal transcribed spacer 2（ITS2） high-throughput sequencing， combined with fungal community diversity analysis and fungal community structure analysis， were used to obtain the fungal flora microbial categories of Aurantii Fructus under the conditions of traditional pressure-shelf fermentation and non-pressure-shelf natural fermentation for 7， 14， 21 d（numbered Y1-Y3 for the former， and numbered F1-F3 for the latter）， respectively. At the same time， the chemical components in the fermentation process were detected by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS）， combined with principal component analysis（PCA）， partial least squares-discriminant analysis（PLS-DA） and compound retention time， parent ions， characteristic fragment ions and other information， the differential compounds between the different fermentation samples were screened and identified. ResultsThe analysis of fungal community diversity showed that the dominant flora did not change at different fermentation time points in the traditional pressure-shelf fermentation method， while in the non-pressure-shelf natural fermentation method， there was a significant difference with the fermentation process， and at the genus level， the dominant genus of samples Y1， Y2， Y3 and F2 was Aspergillus， while the dominant genera of samples F1 and F3 were both Rhizopus. This indicated that the microbial growth environment provided by the traditional fermentation method was more stable， and the microbial community structure was more stable， which was more conducive to the stable and controllable fermentation process and fermented products. A total of 155 compounds were identified by compositional analysis， including 70 flavonoids， 38 coumarins， 10 alkaloids， 34 organic acids and 3 other compounds. After fermentation， two new components of ribalinine and pranferin were produced. Different fermentation conditions also brought about differences in chemical composition， multivariate statistical analysis obtained 26 differential compounds under two different fermentation methods， mainly including flavonoids， organic acids and coumarins. Comprehensively， the microbial community structure of samples fermented by the traditional pressure-shelf method of Aurantii Fructus for 14 d was stable， the species richness was high and the overall content of differential compounds was high， which was the optimal processing condition. ConclusionCompared with non-pressure-shelf natural fermentation， the traditional method has obvious advantages in terms of the stability of the microbial community structure and the content of chemical compounds， and the optimal condition is 14 days of fermentation. This study is helpful to promote the quality stability and fermentation bioavailability of fermented products of Aurantii Fructus， as well as to provide an experimental basis for the further improvement of the quality control methods of this variety.

Objective To investigate the regulatory effect of circular RNA ASAP1(circASAP1)targeting microRNA-4500(miR-4500)on the proliferation and apoptosis of osteosarcoma cells.Methods Human osteosarcoma cells(Saos-2)were selected as the experimental subjects and cul-tured in DMEM medium containing 10％fetal bovine serum while maintaining 5％CO2.The expres-sions of circASAP1 and miR-4500 in osteosarcoma tissues were detected using RT-qPCR.Saos-2 cells were transfected with si-NC(40 nmol/L),si-circASAP1(40 nmol/L),pcDNA(0.4 μg),pcDNA-circASAP1(0.4 μg),miR-NC(40 nmol/L),miR-4500 mimics(40 nmol/L),si-circASAP1+anti-miR-NC(40 nmol/L),and si-circASAP1+anti-miR-4500(40 nmol/L),respectively.Cell proliferation and apoptosis were assessed using CCK-8,colony formation assays,and flow cytometry.The interaction between circASAP1 and miR-4500 was analyzed using a dual-luciferase reporter assay.Results Compared with adjacent non-tumor tissues,the expression of circASAP1 was significantly upregulated,while that of miR-4500 was significantly downregulated in osteosarcoma tissues(P＜0.001).Compared with the si-NC group,the si-circASAP1 group exhibited significantly decreased circASAP1 expression,optical density(OD)values,and the number of cell colonies(P＜0.001).Compared with the si-NC group,the si-circASAP1 group showed significantly upregulated levels of cleaved-caspase3 protein,cleaved-caspase9 protein,and the apoptosis rate(P＜0.001).StarBase search revealed specific binding sequences between miR-4500 and circASAP1.Co-transfection of miR-4500 mimics and WT-circASAP1 significantly reduced the relative luciferase activity of the cells[(0.34±0.03)versus(0.95±0.06),t=27.280,P＜0.001].The expression of miR-4500 in the pcDNA-circASAP1 group was significantly lower than that in the pcDNA group,whereas the ex-pression of miR-4500 in the si-circASAP1 group was significantly higher than that in the si-NC group(P＜0.001).Compared with the miR-NC group,the miR-4500 group exhibited significantly in-creased miR-4500 expression,cleaved-caspase3 protein levels,cleaved-caspase9 protein levels,and the apoptosis rate,while the OD values and the number of colonies significantly decreased(P＜0.001).Compared with the si-circASAP1+anti-miR-NC group,the si-circASAP1+anti-miR-4500 group showed significantly decreased miR-4500 expression,cleaved-caspase3 protein levels,cleaved-caspase9 protein levels,and the apoptosis rate,while the OD values and the number of col-onies significantly increased(P＜0.001).Conclusion The expression of circASAP1 is increased,while that of miR-4500 is decreased in osteosarcoma tissues.Moreover,circASAP1 promotes the proliferation and inhibits the apoptosis of osteosarcoma cells by targeting miR-4500.

Objective To construct and verify a recurrence risk prediction model for patients with malig-nant pleural and peritoneal effusion after deep hyperthermia based on the proportion of peripheral blood CD3+T cell subsets and the expression profile of inflammatory cytokines.Methods A retrospective analysis was conducted on the clinical characteristics,diagnosis and treatment processes of 188 patients with malignant pleural and peritoneal effusion who visited this hospital from September 2023 to May 2024.All patients re-ceived chemotherapy combined with deep hyperthermia and were divided into the non-recurrence group(n=130)and the recurrence group(n=58)based on whether malignant pleural and peritoneal effusion recurred within 3 months after the end of treatment.The differences in general clinical data,conventional tumor mark-ers at the end of treatment,the proportion of CD3+T cell subsets,and the expression profile levels of inflam-matory cytokines between the two groups of patients were compared.The risk factors for recurrence in pa-tients with malignant pleural and peritoneal effusion were screened through univariate and multivariate Logis-tic regression analyses,and a risk prediction model was established.The receiver operating characteristic(ROC)curve was applied to evaluate the effectiveness of this prediction model for the recurrence risk of pa-tients with malignant pleural and peritoneal effusion after chemotherapy combined with deep hyperthermia.Results The age of patients in the recurrence group,the proportion of stage Ⅳ patients,the levels of serum CEA,CA125,TGF-β,and the proportion of CD8+T cells were significantly higher than those in the non-recur-rence group(P＜0.05),while the proportion of CD4+T cells,CD4+/CD8+,and the level of serum IL-10 were significantly lower than those in the non-recurrence group(P＜0.05).The results of univariate and multivari-ate Logistic regression analysis indicated that CD4+T,CD8+T,CD4+/CD8+,IL-10,and TGF-β were all im-portant influencing factors for recurrence after chemotherapy combined with deep hyperthermia in patients with malignant pleural and peritoneal effusion(P＜0.05).The AUC(95％CI)of the predictive model con-structed based on the above influencing factors by ROC curve analysis was 0.708(0.614-0.802),suggesting a high clinical predictive efficacy.Conclusion The predictive model constructed based on T-cell subset counts and serum inflammatory cytokines can effectively predict the recurrence risk of patients with malignant pleu-ral and peritoneal effusion after chemotherapy combined with deep hyperthermia.It has certain clinical value for the therapeutic effect evaluation of patients with malignant pleural and peritoneal effusion and for guiding the optimization of deep hyperthermia regimens.

Objective To explore the distribution of common TCM syndromes and symptoms of insomnia;To prepare for the construction of the theoretical framework and item pool of syndrome diagnosis and efficacy evaluation scale.Methods TCM guideline standards of insomnia,textbooks and journals over the years were retrieved,the information of TCM syndromes,syndrome elements and symptoms was extracted,the guideline textbook and journal database were established,and descriptive statistics,association rules,systematic clustering,factor analysis,potential categories and implicit structure analysis were carried out.Results Totally 116 guide standards and textbooks over the years were included,and 454 articles of journals were included.The high-frequency symptoms accounted for≥3%of the guide textbooks and journal databases were 87 and 79 categories,respectively,and the cumulative proportion was 87.48%and 87.75%,respectively.According to the analysis results,five common TCM syndromes and their characteristic symptom classification of insomnia were finally deduced.According to the frequency/person time distribution,they were heart and spleen deficiency syndrome,yin deficiency and fire hyperactivity syndrome,liver fire disturbing heart syndrome,phlegm heat disturbing heart syndrome,heart and gallbladder qi deficiency syndrome.Conclusion There are five common TCM syndromes of insomnia,and the characteristic symptoms of each TCM syndrome provide a reference source for the theoretical framework of syndrome diagnosis and efficacy evaluation scale and the establishment of item pool.

Kleine-Levin syndrome (KLS) is a rare, recurrent-remitting sleep disorder characterized by periodic hypersomnia, hyperphagia, and hypersexuality, accompanied by cognitive dysfunction, perceptual disturbances, mood changes, or psychiatric abnormalities. Patients typically experience recurrent episodes, with hypersomnic periods lasting from several days to weeks. Most KLS patients exhibit normal functions during the intermission period, while early-onset KLS cases in adolescents are rare, often presenting with frequent episodes and complex symptoms, which can easily lead to misdiagnosis. Therefore, this case report analyzed the detailed medical history, laboratory findings, differential diagnosis, and treatment of a teenage patient with KLS, along with a review of the literature. This case aims to provide a deeper understanding of the complex symptoms and diagnostic challenges associated with early-onset KLS in adolescents.

Objective To explore the distribution of common TCM syndromes and symptoms of insomnia;To prepare for the construction of the theoretical framework and item pool of syndrome diagnosis and efficacy evaluation scale.Methods TCM guideline standards of insomnia,textbooks and journals over the years were retrieved,the information of TCM syndromes,syndrome elements and symptoms was extracted,the guideline textbook and journal database were established,and descriptive statistics,association rules,systematic clustering,factor analysis,potential categories and implicit structure analysis were carried out.Results Totally 116 guide standards and textbooks over the years were included,and 454 articles of journals were included.The high-frequency symptoms accounted for≥3%of the guide textbooks and journal databases were 87 and 79 categories,respectively,and the cumulative proportion was 87.48%and 87.75%,respectively.According to the analysis results,five common TCM syndromes and their characteristic symptom classification of insomnia were finally deduced.According to the frequency/person time distribution,they were heart and spleen deficiency syndrome,yin deficiency and fire hyperactivity syndrome,liver fire disturbing heart syndrome,phlegm heat disturbing heart syndrome,heart and gallbladder qi deficiency syndrome.Conclusion There are five common TCM syndromes of insomnia,and the characteristic symptoms of each TCM syndrome provide a reference source for the theoretical framework of syndrome diagnosis and efficacy evaluation scale and the establishment of item pool.

Kleine-Levin syndrome (KLS) is a rare, recurrent-remitting sleep disorder characterized by periodic hypersomnia, hyperphagia, and hypersexuality, accompanied by cognitive dysfunction, perceptual disturbances, mood changes, or psychiatric abnormalities. Patients typically experience recurrent episodes, with hypersomnic periods lasting from several days to weeks. Most KLS patients exhibit normal functions during the intermission period, while early-onset KLS cases in adolescents are rare, often presenting with frequent episodes and complex symptoms, which can easily lead to misdiagnosis. Therefore, this case report analyzed the detailed medical history, laboratory findings, differential diagnosis, and treatment of a teenage patient with KLS, along with a review of the literature. This case aims to provide a deeper understanding of the complex symptoms and diagnostic challenges associated with early-onset KLS in adolescents.

OBJECTIVE To investigate the use of drugs and the development of pharmaceutical care in the tight medical alliance (shorted for "medical alliance") of Wanzhou District of Chongqing,and provide reference for the further construction of the medical alliance.METHODS A survey form was designed and distributed to 21 constituent units (5 leading units and 16 member units) of 5 medical alliances in Wanzhou District of Chongqing.The statistical analysis was conducted in aspects of basic drug allocation and use,pharmaceutical personnel team construction,the development of pharmaceutical care,and rational use of antibiotics.RESULTS Among the 21 constituent units,4 leading units and 14 member units achieved the target for the proportion of essential drug procurement varieties,with a total compliance rate of 85.71％;4 leading units and 13 member units achieved the target for the proportion of national essential drug allocation and usage amount,with a total compliance rate of 80.95％.The proportions of personnel with doctoral degrees in the 5 leading units and 16 member units were 1.71％ and 0 respectively,and the proportions of personnel with senior professional titles were 8.56％ and 1.63％,respectively.A total of 5 pharmacy or pharmaceutical combined outpatient clinics were set up in the 21 medical alliance units,and 5 clinical pharmacy information service platforms were established;all 5 leading units were able to regularly carry out clinical pharmacy projects,while only 4 out of 16 member units had conducted medical order review and evaluation.The proportions of irrational use of antibiotics in outpatient prescriptions and inpatient medical records of the 16 member units (4.81％,5.21％) were significantly higher than those of the 5 leading units (2.80％,4.00％).CONCLUSIONS The allocation and usage of national essential drugs in 21 constituent units from Wanzhou District of Chongqing are both in good standing.However,the data on the allocation of pharmaceutical professionals and the number,qualifications,and job titles of clinical pharmacists in member units are generally low.Moreover,the pharmaceutical service projects and service quality in member units need to be further improved.

Objectives:To analyze the clinical prognosis stratification of fetal heart disease(FHD)and the mid-term follow-up results following integrated prenatal and postnatal management. Methods:Present retrospective analysis was performed on 817 fetuses diagnosed with FHD by fetal echocardiography in Fuwai Hospital,Chinese Academy of Medical Sciences(Fuwai Hospital)from the 8th February 2018 to 30th April 2022.According to the Chinese expert consensus on fetal prognosis score of congenital heart disease(CHD)and Fuwai Hospital Pediatric Center score,FHD was divided into grade Ⅰ(0 point),grade Ⅱ(1-3 points),grade Ⅲ(4-6 points)and grade Ⅳ(7-9 points).For FHD fetuses that need to be treated in the neonatal period,the whole closed-loop management and treatment were completed,including pregnancy,birth,neonatal monitoring,surgery,and follow-up.The clinical data and mid-term follow-up results of FHD fetuses were analyzed. Results:A total of 752 fetuses completed the mid-term follow-up.The mean follow-up time was(18.5±2.3)months.There were 111 cases(14.8%)of FHD grade Ⅰ,251 cases(33.4%)of FHD grade Ⅱ,275 cases(33.6%)of FHD grade Ⅲ and 115 cases(15.3%)of FHD grade Ⅳ.There were 393 cases(52.2%)of fetal preservation,1 case(0.2%)of intrauterine death,and 358 cases(47.6%)of induced abortion.Compared with the induced fetus,the pregnant women in the reserved fetus group were older,the gestational age was longer,the rate of receiving noninvasive DNA and amniocentesis was lower,the proportion of chromosomal abnormalities or genetic abnormalities was smaller,and the FHD prognostic score was lower(all P<0.05).One case(0.9%)of FHD grade Ⅰ fetus experienced intrauterine fetal death,and 110 cases(99.1%)were born and recovered.164 cases(65.3%)of grade Ⅱ fetuses were born,and all of them were cured by surgery or spontaneously.118 cases(42.9%)of grade Ⅲ fetuses were born,117 cases completed biventricular radical operation,and 1 case completed two-stage operation waiting for the second stage biventricular radical operation.One case(0.9%)of grade Ⅳ was born and completed the two-stage bidirectional Glenn procedure.Among the induced FHD grade Ⅱ fetuses,8(9.2%)had abnormal chromosomal or genetic testing results,and 8(9.2%)had other organ abnormalities.Among the induced FHD grade Ⅲ fetuses,5 cases(3.2%)had chromosomal abnormalities,5 cases(3.2%)had genetic abnormalities,and 9 cases(5.7%)had other organ abnormalities.There was only one case(0.9%)of FHD Ⅳ with hypoplastic left heart syndrome,which was retained as one of the twins and was born successfully.Norwood one-stage procedure was performed in the neonatal period,and two-stage bidirectional Greene procedure was performed at 6 months after birth.Other fetuses were induced following the choice of maternity and their family members. Conclusions:Hierarchical management based on clinical prognosis is helpful to scientifically guide the integrated treatment of FHD fetuses and improve the success rate of treatment of FHD fetuses with good prognosis.