ObjectiveThis study aims to enhance of the farnesyl pyrophosphate（FPP） pool in Saccharomyces cerevisiae by heterologously expressing different farnesyl diphosphate synthases（FPSs） from various plants， thereby increasing the production of terpenoid compounds by the engineered yeast. MethodsRNA from mixed samples of roots， stems， and leaves of seven plants including Arabidopsis thaliana， Rosa rugosa， Artemisia annua， Centella asiatica， Humulus lupulus， Medicago sativa， and Panax ginseng was extracted by column chromatography and reverse transcribed into the first strand of complementary DNA（cDNA）， and based on the transcriptome data of the seven species of plants， sequence-specific primers were designed for CaFPS， RrFPS， MsFPS， HiFPS， PgFPS， AtFPS， and AaFPS， the full-length of the genes was cloned， and the genes were analyzed for bioinformatics in order to construct a pESC yeast shuttle vector. These seven plant-derived FPSs were further heterologously expressed in the previous constructed β-elemene-producing yeast， and the yield of β-elemene was indicated for their catalytic acivities. ResultsThe coding sequences of CaFPS， RrFPS， MsFPS， HiFPS， PgFPS， AtFPS， and AaFPS were all of 1 021 bp in length and encoding 301 amino acids， all of which were similarly related to the endogenous FPS-encoding gene（ERG20） in S. cerevisiae. After heterologous expression， RrFPS was identified as the most effective in catalyzing the synthesis of FPP from isopentenyl pyrophosphate（IPP） and dimethylallyl pyrophosphate（DMAPP）. Compared to the control strains， the RrFPS overexpressed yeast strains YB-1-Rr and YB-3-Rr increased the production of β-elemene by 231.25% and 189.3%， respectively. ConclusionBy comparing the functions of FPS-encoding genes from seven different plant sources， it is determined that the protein encoded by the RrFPS from R. rugosa has the best catalytic ability， which can provide key genetic elements for the construction of engineered yeast strain constructs with high terpenoid production.

Objective:To discuss the regulatory effect of berberine(BBR)on fatty acids in the human glioma T98G cells and its effect on the cell proliferation,migration,and invasion,and to clarify its potential mechanism.Methods:The T98G cells at logarithmic growth phase were divided into control group and different concentrations(25,50,and 100 mg·L-1)of BBR groups.Cell wound healing assay was used to detect the migration rates of the cells in various groups;Transwell chamber assay was used to detect the invasion rates of the cells in various groups.The T98G cells at logarithmic growth phase were divided into control group and 100 mg·L-1 BBR group,and Mass spectrometry was used to detect the fatty acid contents in the cells in two groups.The T98G cells at logarithmic growth phase were divided into control group and different concentrations(50,100,and 150 mg·L-1)of BBR groups;Western blotting method was used to detect the expression levels of phosphatidylinositol 3-kinase(PI3K),phosphorylated PI3K(p-PI3K),protein kinase B(AKT),phosphorylated AKT(p-AKT),sterol regulatory element-binding protein 1(SREBP-1),and fatty acid synthase(FASN)in the cells in various groups.The expression of FASN was suppressed by gene silencing technology,and the T98G cells at logarithmic growth phase were divided into control group,shFASN1 group,and shFASN2 group.Western blotting method was used to detect the expression levels of FASN protein in the cells in various groups;clone formation assay was used to detect the clone formation of the cells in various groups;cell wound healing assay was used to detect the migration rates of the cells in various groups.Results:Compared with control group,the migration rates and invasion rates of the cells in different concentrations of BBR groups were decreased in a concentration-dependent manner(P<0.01),and the fatty acid content in the cells in 100 mg·L-1 BBR group was significantly decreased(P<0.01).Compared with control group,the expression levels of p-PI3K,p-AKT,SREBP-1,and FASN proteins in the cells in 150 mg·L-1 BBR group were significantly decreased(P<0.05 or P<0.01),and the expression level of SREBP-1 protein in the cells in 100 and 150 mg·L-1 BBR groups were significantly decreased(P<0.01).After suppression of FASN expression,compared with control group,the expression levels of FASN protein in the cells in shFASN1 and shFASN2 groups were significantly decreased(P<0.01),and the expression level of FASN protein in the cells in shFASN2 group was lower than that in shFASN1 group(P<0.05);compared with control group,the numbers of clone formation and migration rates of the cells in shFASN1 and shFASN2 groups were significantly decreased(P<0.01),and the migration rate of the cells in shFASN2 group was significantly lower than that in shFASN1 group(P<0.05).Conclusion:BBR interferes with fatty acid synthesis in the glioma T98G cells by reducing the expression of the PI3K/AKT/SREBP-1/FASN pathway related proteins,and decrease their migration and invasion capabilities.

Objective:To quantitatively analyze the plaque components of carotid artery through energy spectrum computed tomography angiography(CTA),and to measure the blood flow perfusion in the blood-supply area of carotid artery through CT perfusion(CTP),so as to explore the relationship among plaque component,the degree of luminal stenosis and cerebral blood flow perfusion.Methods:A total of 68 patients with unilateral plaques of carotid artery and severe vascular stenosis who were screened and diagnosed by ultrasound and CTA in Xiyuan Hospital from December 2017 to July 2019 were selected,and all patients underwent CTA examination and CTP examination.North American symptomatic carotid endarterectomy test(NASCET)method was used to measure the degree of carotid stenosis.The GE AW 4.7 post-process workstation was used to conduct analyses of energy spectrum and cerebral perfusion for the plaque component.And then,the slope of energy spectrum curve and the effective atomic number were obtained.At the same time,the cerebral blood volume(CBV),cerebral blood flow(CBF),time to peak(TTP)and mean transit time(MTT)of contrast agent in blood-supplying area of anterior cerebral artery(ACA)and middle cerebral artery(MCA)at the side of lesion were measured.Results:A total of 68 measured plaques of 68 patients met the condition,including 44 vulnerable plaques(including lipid plaques and mixed plaques)and 24 stable plaques(fibrous plaques).The average slopes of the energy spectrum curves of vulnerable plaque and stable plaque were respectively 0.45±0.45 and 1.15±0.39,and the differences were significant(t=2.413,P<0.05).The averagely effective atomic numbers of vulnerable plaques and stable plaques were respectively 7.21±1.06 and 8.01±0.63,and the difference were significant(t=2.548,P<0.05).The average TTP values of the ACA at the side of lesion of vulnerable plaques and stable plaques were respectively(12.20±1.61)S and(13.59±2.79)S,and the difference was significant(t=-2.607,P<0.05).The mean MTT values of the ACA at the side of lesion of vulnerable plaques and stable plaques were respectively(5.07±1.66)S and(6.09±2.19)S,and the difference was significant(t=-2.177,P<0.05).The degree of vascular stenosis at the side of lesion was positively correlated with TTP and MTT in blood-supplying area of middle cerebral artery(MCA)at the side of lesion(r=0.537,0.465,P<0.05),and that was negatively correlated with CBF values in blood-supplying areas of ACA and MCA at the side of lesion(r=-0.281,-0.569,P<0.05),respectively.The slope of the energy spectrum curve of carotid plaque was positively correlated with the TTP values in blood-supplying areas of ACA and MCA at the side of lesion(r=0.242,0.246,P<0.05),respectively.Conclusion:CT spectral imaging can quantitatively analyze the displayed components of carotid atherosclerotic plaque,and the degree of vascular stenosis can affect the blood flow perfusion of cerebral tissue,and the delays of TTP and MTT are more easily caused by vulnerable plaque,and the TTP of them is more sensitivity.

There are a total of 16 couplet medicines of Paeoniae Radix Alba and Glycyrrhizae Radix et Rhizoma in the book FU Qingzhu Nüke, covering a wide range of diseases such as leukorrheal diseases, menopathy, pregnancy, and puerperal diseases, and there is a fine sense of the dosage, processing, and proportion of the couplet medicines. Through analyzing the cases of Paeoniae Radix Alba and Glycyrrhizae Radix et Rhizoma couplet medicines and the characteristics of dosage, processing, and proportion, we conclude that Paeoniae Radix Alba and Glycyrrhizae Radix et Rhizoma couplet medicines are mainly as followed: smoothing liver and strengthening spleen in treating leukorrheal diseases to remove dampness and stop leucorrhoea; regulating liver and tonifying spleen in menopathy to regulate menstruation and relieve pain; nourishing blood and benefiting qi in pregnancy to lower adverse qi and tranquilize fetus to prevent miscarriage; regulating and tonifying qi and blood in puerperal diseases to eliminate pathogenic factors and promote lactation; suppressing hyperactive liver for descending adverse qi, and relieving spasm and pain in cases of miscarriage due to rage. In terms of dosage, Paeoniae Radix Alba is mainly used for three to five qian, and Glycyrrhizae Radix et Rhizoma is primarily used for one qian, and the dosage of the two medicinals is adjusted according to the degree of primary and secondary liver stagnation and spleen deficiency. In terms of processing, wine Paeoniae Radix Alba and raw Glycyrrhizae Radix et Rhizoma are primarily used. Stir-frying with wine can help Paeoniae Radix Alba nourish blood and promote blood circulation, tonifying without stagnation, and it is used in most of the diseases caused by liver qi stagnation or qi and blood deficiency. Glycyrrhizae Radix et Rhizoma used in raw is tonifying without causing stagnation, and it can also have the effect of purging fire. In terms of proportion, the ratio of Paeoniae Radix Alba to Glycyrrhizae Radix et Rhizoma is 5∶1 for liver stagnation restraining spleen, 3∶1 for qi and blood deficiency, and 1∶1 for obvious fire-heat. This paper analyses the application of Paeoniae Radix Alba and Glycyrrhizae Radix et Rhizoma couplet medicines in FU Qingzhu Nüke, aiming to deeply study and inherit the academic thought of FU Qingzhu, and to provide new ideas and method for the precise application of Paeoniae Radix Alba and Glycyrrhizae Radix et Rhizoma couplet medicines in clinical practice and researches.

Objective To investigate the mechanism by which EFHD2 affects the occurrence and progression of breast cancer based on the NOX4/ROS signaling pathway.Methods Cells were divided into an NC-shRNA group and EFHD2-shRNA group.A lentiviral vector for EFHD2 silencing and a control vector were constructed and used to transfect MDA-MB-23 and MCF-7 breast cancer cells.The transfection efficiency was verified by qRT-PCR.A CCK8 assay was used to detect cell proliferation activity.A plate cloning assay was employed to measure cell colony formation ability.A scratch test was used to detect cell migration,and a Transwell assay used to assess cell invasion.Flow cytometry was applied to detect apoptosis and ROS levels.qRT-PCR was used to analyze the mRNA expression of GLUT1,PDK1,PFK1,PKM2,PDH,and LDH,while Western blot was applied to detect the expression of Cleaved caspase-3,MMP-2,and NOX4 proteins.Results Compared with the NC-shRNA group,the EFHD2-shRNA group's EFHD2 expression was significantly decreased and its cell survival and colony formation ability were weakened.The apoptosis rate and the expression of the pro-apoptotic protein Cleaved caspase-3 increased.The cell migration distance was shortened,while the number of invading cells and the expression of MMP-2,which promotes migration and invasion,were decreased.The levels of lactic acid and GLUT1,PDK1,PFK1,PKM2,and LDH decreased,while the levels of ATP and PDH increased.Streaming result showed that ROS levels were reduced and NOX4 protein was down-regulated after silencing EFHD2.Conclusions EFHD2 inhibits ROS production by regulating the NOX4/ROS signaling pathway,causing lactic acid and glucose accumulation,promoting the apoptosis of breast cancer cells,and inhibiting cell proliferation,migration,and invasion.

Immune checkpoint inhibitors (ICIs) have demonstrated unparalleled clinical responses and revolutionized the paradigm of tumor treatment, while substantial patients remain unresponsive or develop resistance to ICIs as a single agent, which is traceable to cellular metabolic dysfunction. Although dysregulated metabolism has long been adjudged as a hallmark of tumor, it is now increasingly accepted that metabolic reprogramming is not exclusive to tumor cells but is also characteristic of immunocytes. Correspondingly, people used to pay more attention to the effect of tumor cell metabolism on immunocytes, but in practice immunocytes interact intimately with their own metabolic function in a way that has never been realized before during their activation and differentiation, which opens up a whole new frontier called immunometabolism. The metabolic intervention for tumor-infiltrating immunocytes could offer fresh opportunities to break the resistance and ameliorate existing ICI immunotherapy, whose crux might be to ascertain synergistic combinations of metabolic intervention with ICIs to reap synergic benefits and facilitate an adjusted anti-tumor immune response. Herein, we elaborate potential mechanisms underlying immunotherapy resistance from a novel dimension of metabolic reprogramming in diverse tumor-infiltrating immunocytes, and related metabolic intervention in the hope of offering a reference for targeting metabolic vulnerabilities to circumvent immunotherapeutic resistance.
Humans ; Neoplasms/pathology* ; Immunotherapy/methods* ; Immune Checkpoint Inhibitors/therapeutic use*

Purpose/Significance To explore the characteristics and clinical significance of differentially expressed genes closely re-lated to HPV E6/E7 by using bioinformatics.Method/Process The cervical tissue and clinical information of cervical cancer in TCGA and GTEx of UCSC are used as the training set.The expression profile chip GSE63514 related to cervical cancer in GEO is used as the validation set.Firstly,the limma package of R software is used to screen DEGs of tumor and normal samples,and Venn map of genes re-lated to E6/E7 protein in MigDB is made.Survival analysis is performed by survival kit and verified by ROC and protein expression lev-els.Secondly,key genes are obtained by copy number variation and methylation correlation.Finally,the specific co-expression network is constructed and enrichment analysis and immune infiltration analysis are performed.Result/Conclusion There are 101 differentially expressed genes related to HPV E6/E7 protein,and three genes are found to have significance after screening,namely E2F1,MCM4 and PCNA.At the same time,it is found that the genes in the specific coexpression network are significantly enriched in the DNA replication and chromosome organization pathways.Immune correlation analysis shows that key genes are significantly associated with CD4 T cells,B cells and neutrophils.DNA replication,chromosome organization,etc.,are the molecular mechanisms and key genes significantly related to the development of cervical squamous cell carcinoma and HPV E6/E7 encoded proteins.

Objective:To retrospectively summarize disease characteristics and the clinical experience of minimally invasive endoscopy in the treatment of upper urinary tract obstruction caused by ureteropelvic encrusted inflammatory disease.Methods:Three patients with bilateral ureteropelvic encrusted inflammatory disease admitted to our hospital from March 2018 to July 2021 were involved. Case 1, male, 45 years old, admitted due to bilateral hydronephrosis for 5 months. The preoperative diagnosis were bilateral ureteropelvic stones (encrustation), right ureteral atresia, left ureteral stenosis, and systemic vasculitis. Left double J tube insertion and right nephrostomy were performed in another hospital. We conducted antegrade percutaneous nephroscopy combined with retrograde ureteroscopy surgery and assisted balloon dilation to treat bilateral lesions stage by stage. Case 2, Male, 12 years old, admitted due to bilateral abdominal pain for 6 weeks. The preoperative diagnosis were bilateral ureteral stones, bilateral hydronephrosis, and dermatomyositis. After the failure of double J tube insertion in another hospital, double nephrostomy was performed instead. We performed left percutaneous nephroscopy and right percutaneous nephroscopy combined with ureteroscopy for the treatment of bilateral lesions. Case 3, female, 32 years old, was admitted because of pain in the left lower back and abdomen for over 6 months. The preoperative diagnosis were bilateral ureteral stones, bilateral ureteral stenosis, and dermatomyositis. She underwent three times of ESWL and once URS before. We performed ureteroscopic surgery for bilateral lesions. During the surgery, various degrees of crusting in the renal pelvis or ureter were observed in all 3 cases, and the lesions were removed using pneumatic lithotripsy combined with forceps or baskets. After surgery, oral antibiotics were continuously used for 1-3 months. The efficacy and prognosis were evaluated based on the follow-up of urine, imaging, and endoscopic examinations at 3, 6, and 12 months after surgery.Results:All 3 surgeries were successfully completed. At 3, 6, and 12 months after surgery, follow-up CT showed no crusting in the left ureter, and endoscopy showed good mucosal wound healing and unobstructed lumen in case 1. There were still some crusting lesions and lumen stenosis in the right renal pelvis, and the right ureter reconstruction surgery was ultimately performed. There were no crusting on both sides and the urinary tract was unobstructed after 3, 6, and 12 months of follow-up in case 2 and case 3. Postoperative pathological examination showed chronic inflammation of urothelial mucosal tissue, small pieces of proliferative fibrous tissue with peripheral calcification. Calcification layer composition analysis showed magnesium ammonium phosphate and carbonate apatite. No related complications occurred in case 2 and case 3.Conclusions:Urothelial crusted inflammatory disease is rare clinically, and the diagnosis and treatment strategies are rarely reported domestically and internationally. Preoperative imaging examination, intraoperative findings and postoperative pathology or calcification composition analysis are of instruction for the diagnosis and treatment of this disease. Minimally invasive endoscopy treatment for upper urinary tract obstruction caused by ureteropelvic encrusted inflammatory disease has a good effect. Long-term efficacy and other adjuvant treatment need long-term follow-up and clinical practice.

Objective:To summarize the preliminary clinical experience of utilizing ureteral balloon dilation catheter in the treatment of "difficult ureter" during ureteroscopic lithotripsy, and to discuss the efficacy and safety of the technique.Methods:Clinical data of 28 patients (30 sides) with upper urinary tract calculi admitted to Beijing Tsinghua Changgung Hospital Affiliated to Tsinghua University from April 2021 to July 2022 were retrospectively analyzed. There were 23 males (82.1%) and 5 females (17.9%), with age of (51.5±13.6) years. Among the 30 sides, 20 (66.7%) on the left and 10(33.3%) were on the right. Calculi were either located in the renal pelvis or calyxes in 7 sides (23.3%), upper ureter in 17 sides (56.7%), and lower ureter in 6 sides (20.0%). The maximum diameter of the stones was (9.4±4.2)mm, and 23 sides (76.7%) were combined with hydronephrosis before surgery. When "difficult ureter" was encountered during the procedure, that is, it was difficult to insert ureteroscope or ureteral access sheath (UAS) due to small ureteral lumen, balloon catheter was used for dilation in the first stage, in which the balloon diameter was 4 mm on 22 sides and 5mm on 8 sides. The instrument was retrogradely inserted through the working channel of F8 semi-rigid ureteroscope, and the small site of the ureteral lumen was dilated under direct endoscopic view. After a single dilation, the balloon catheter was withdrawn, and the effect of dilation was evaluated by semi-rigid ureteroscopy to determine whether to proceed with the following procedures. The intraoperative data were recorded, including surgical method, stage of "difficult ureter" occurred, site of the small part of the ureter, related data of utilizing ureteral dilatation balloon catheter, grade of ureteral injury after dilatation (according to the 0-4 grading classification of endoscopic ureteral injuries), total operation time, balloon catheter-related adverse events, stone-free rate, and time of removing ureteral stents.Results:Among the 30 sides, 29 (96.7%) had difficulty in the stage of ureteroscope insertion, and 1(3.3%) had difficulty in the stage of UAS insertion. A total of 37 small sites of ureter were involved, including 18 in the intramural segment, 10 in the lower part, 2 in the middle part, and 7 in the upper part. Each site was dilated once with a median time of 3 (0.5, 5.0) minutes and a median maximum balloon pressure of 1 215.9(1 215.9, 1 443.9)kPa[12.0(12.0, 14.3)atm]. There were 28 sites of grade Ⅰ injury, 8 sites of grade Ⅱinjury, and 1 site of grade Ⅲinjury. The total duration of unilateral procedure was (73.4±30.3) min. Ureteroscope or UAS insertion was successful in 28 sides(93.3%) after balloon dilation, and failed in 2 sides(6.7%), both of which were in the stage of inserting ureteroscope and ureteral stent was indwelled for the second-stage procedures. On the first day after surgery, the hemoglobin level was (134.1±12.9)g/L, which was significantly different from the preoperative parameters ( P<0.01), and serum creatinine level was (86.7±23.2)μmol/L, which showed no significant difference from the preoperative one ( P=0.263). The primary stone-free rate was 92.9% (26/28), and the total postoperative complication rate was 13.3% (4/30), including 3 of grade Ⅰ (lateral lower abdominal pain requiring additional analgesic drugs) and 1 of grade Ⅱ (postoperative hematuria requiring intravenous hemostatic drugs). Follow-up was conducted for 3 months. All of the 28 successful sides had their ureteral stents removed before the last follow-up, and the time of removal was (36.9±11.5) days. No hydronephrosis was found in the ipsilateral kidney by ultrasound 3 months after operation. Conclusions:Balloon dilation technique showed good efficacy and safety in the treatment of "difficult ureter" during ureteroscopic lithotripsy.

Volatile oil microemulsion gel of traditional Chinese medicine has the dual characteristics of microemulsion and gel， which can improve the solubility and stability of drugs， promote drug absorption and achieve long-term sustained release effect. In recent years， it has been widely used in clinical diseases such as internal medicine， gynecology and pediatrics. In this paper， the characteristics， prescription design， molding process and quality evaluation of volatile oil microemulsion gel of traditional Chinese medicine were reviewed by searching relevant literature. The results showed that the volatile oil microemulsion gel had the characteristics of improving the stability of the drug， increasing the transdermal penetration of the drug， strong adhesion， easy to wash， convenient preparation， etc. The prescription design consists of oil phase， surfactant， cosurfactant and gel matrix. The molding process includes the molding process of microemulsion and the molding process of gel， and the formation of microemulsion mainly uses the pseudo-ternary phase diagram method. The quality evaluation can be divided into 2 parts： volatile oil of traditional Chinese medicine and microemulsion gel. The quality evaluation of volatile oil can be studied qualitatively and quantitatively. Microemulsion gel should meet the relevant requirements and be tested for in vitro release and transdermal absorption.