BACKGROUND:Solute carrier family 1 member 5(SLC1A5)plays a potential role in a variety of diseases,but the exact mechanism of action is unclear.The construction of stable SLC1A5 overexpression and knockdown cell models can provide a powerful experimental tool for in-depth study of the exact role and mechanism of SLC1A5 in diseases and the discovery of potential therapeutic targets. OBJECTIVE:To construct lentiviral vectors for overexpression and knockdown of mouse SLC1A5 and establish stable transfected RAW264.7 cell lines,so as to provide an experimental foundation for further investigation of the role of SLC1A5 in inflammation. METHODS:Primers were designed and synthesized based on the SLC1A5 gene sequence,and the gene segment was amplified using polymerase chain reaction.Subsequently,the target gene segment was directionally inserted into the GV492 vector plasmid,which had been digested with AgeI/NheI enzymes,to construct recombinant lentiviral plasmids.Positive clones were further selected,and their sequences were confirmed.The pHelper1.0 plasmid vector and pHelper2.0 plasmid vector,along with the target plasmid vector,was co-cultured with 293T cells for transfection,resulting in the production and titration of lentiviral stocks.Furthermore,RAW264.7 cells were cultured in vitro,and the working concentration of puromycin was determined.Lentiviruses were separately co-cultured with RAW264.7 cells,and transfection efficiency was determined by measuring fluorescence intensity.Stable transfected cells were selected using puromycin,and real-time fluorescence quantitative PCR and western blot assay were employed to assess the gene and protein expression levels of SLC1A5 in stably transfected cell lines. RESULTS AND CONCLUSION:(1)Sequencing results indicated a perfect match between the sequencing and target sequences,confirming the successful construction of recombinant lentiviral vectors.(2)The titer for the overexpression SLC1A5 lentivirus was 1×109 TU/mL,while the titer for the knockdown SLC1A5 lentivirus was 3×109 TU/mL.(3)The working concentration of puromycin for RAW264.7 cells was determined to be 3 μg/mL.(4)The optimal conditions for transfecting RAW264.7 cells with overexpression/knockdown expression of SLC1A5 lentivirus involved the use of HiTransG P transfection enhancer with a multiplicity of infection value of 50.(5)A significant upregulation of the gene and protein expression levels of SLC1A5 was detected in cell lines stably overexpressing SLC1A5,while gene and protein expression levels of SLC1A5 were significantly decreased in the knockdown stable cell lines.These findings indicate that lentiviral vectors for mouse SLC1A5 overexpression and knockdown have been successfully constructed and a stably transfected RAW264.7 cell line has been obtained.

A method for the pretreatment and qualitative detection of 26 trace cathinone new psychoactive substances in wastewater was established and applied in actual wastewater cases. The effluent samples were eluted on the Oasis PRiME HLB solid phase extraction column by ultra-pure water drenching and methanol solution, then dried with nitrogen at 40 ℃, and finally re-dissolved with 0.1% formic acid-acetonitrile solution (95∶5), and detected by liquid chromatography-tandem mass spectrometry, The effluent sample was determined by high-performance liquid chromatography-Tandem mass spectrometry (HPLC-MS/MS) using selected reaction monitoring (SRM) mode and separated on chromatographic column UPLC BEH C18(100 mm×2.1 mm, 1.7 μm） at 35 ℃ with a mobile phase consisting of acetonitrile-0.1% formic acid in aqueous solution gradient elution. After methodological validation, the lower quantification of 26 cathinone new psychoactive substances could reach 1.50−3.00 ng/L. Among these, 21 analytes fell within the concentration range of 1.50−375.0 ng/L, while 5 were detected in the range of 3.00−750.0 ng/L, the correlation coefficient was 0.99, within-and between-batch precision was less than 7.71% and 13.91%, respectively, and the extraction recoveries were higher than 92.64% . The method is simple, accurate, and sensitive, and can be used for cathinone detection and abuse monitoring.

Purpose To explore the value of ultrasonography in distinguishing periductal mastitis(PDM)from granulomatous lobular mastitis(GLM)and summarize the sonographic features of non-puerperal mastitis(NPM).Materials and Methods The ultrasonographic findings of 134 NPM(84 PDM,50 GLM)patients treated in the Second Hospital of Lanzhou University from July 2016 to June 2021 were retrospectively analyzed.Comparing PDM and GLM sonograms,the difference of lesion number,lesion side,lesion orientation,distance from the nipple,long diameter,thick diameter,aspect ratio,boundary,edge,edge,shape,internal echo,peripheral high echo halo,rear echo,calcification,internal blood flow,ipsilateral axillary lymph node enlargement,and summarize the characteristics of NPM according to the lesion morphology and internal echo.Results There was no statistical difference between PDM and GLM(P＞0.05).But the probability of PDM combined with ipsilateral axillary lymph node enlargement was slightly higher than that of GLM(x2=4.209,P=0.040).The ultrasonography of 134 cases of NPM lesions was divided into 7 types according to the morphology and echo changes.The abscess type was more common in GLM than in PDM(x2=4.928,P=0.026).Conclusion There is no significant difference between PDM and GLM.In the case that PDM and GLM cannot be distinguished clinically and radiologically,it is recommended to perform puncture biopsy to determine the pathological type before treatment,which may be more conducive to obtaining the best prognosis for patients.In addition,the classification of NPM into 7 types is helpful for sonographers to grasp the ultrasonographic characteristics of NPM to diagnose NPM early.

OBJECTIVE: To establish the finite element model of varus-type ankle arthritis and to implement the finite element mechanical analysis of different correction models for tibial anterior surface angle (TAS) in supramalleolar osteotomy. METHODS: A female patient with left varus-type ankle arthritis (Takakura stage Ⅱ, TAS 78°) was taken as the study object. Based on the CT data, the three-dimensional model of varus-type ankle arthritis (TAS 78°) and different TAS correction models [normal (TAS 89°), 5° valgus (TAS 94°), and 10° valgus (TAS 99°)] were created by software Mimics 21.0, Geomagic Wrap 2021, Solidworks 2017, and Workbench 17.0. The 290 N vertical downward force was applied to the upper surface of the tibia and 60 N vertical downward force to the upper surface of the fibula. Von Mises stress distribution and stress peak were calculated. RESULTS: The finite element model of normal TAS was basically consistent with biomechanics of the foot. According to biomechanical analysis, the maximum stress of the varus model appeared in the medial tibiotalar joint surface and the medial part of the top tibiotalar joint surface. The stress distribution of talofibular joint surface and the lateral part of the top tibiotalar joint surface were uniform. In the normal model, the stress distributions of the talofibular joint surface and the tibiotalar joint surface were uniform, and no obvious stress concentration was observed. The maximum stress in the 5° valgus model appeared at the posterior part of the talofibular joint surface and the lateral part of the top tibiotalar joint surface. The stress distribution of medial tibiotalar joint surface was uniform. The maximum stress of the 10° valgus model appeared at the posterior part of the talofibular joint surface and the lateral part of the top tibiotalar joint surface. The stress on the medial tibiotalar joint surface increased. CONCLUSION With the increase of valgus, the stress of ankle joint gradually shift outwards, and the stress concentration tends to appear. There was no obvious obstruction of fibula with 10° TAS correction. However, when TAS correction exceeds 10° and continues to increase, the obstruction effect of fibula becomes increasingly significant.
Humans ; Female ; Tibia/surgery* ; Finite Element Analysis ; Ankle ; Arthritis ; Fibula/surgery* ; Ankle Joint/surgery*

AIM: To study the inhibitory effect of Xihuang Pill on H

Constitutively activated G proteins caused by specific mutations mediate the development of multiple malignancies. The mutated Gαq/11 are perceived as oncogenic drivers in the vast majority of uveal melanoma (UM) cases, making directly targeting Gαq/11 to be a promising strategy for combating UM. Herein, we report the optimization of imidazopiperazine derivatives as Gαq/11 inhibitors, and identified GQ262 with improved Gαq/11 inhibitory activity and drug-like properties. GQ262 efficiently blocked UM cell proliferation and migration in vitro. Analysis of the apoptosis-related proteins, extracellular signal-regulated kinase (ERK), and yes-associated protein (YAP) demonstrated that GQ262 distinctly induced UM cells apoptosis and disrupted the downstream effectors by targeting Gαq/11 directly. Significantly, GQ262 showed outstanding antitumor efficacy in vivo with good safety at the testing dose. Collectively, our findings along with the favorable pharmacokinetics of GQ262 revealed that directly targeting Gαq/11 may be an efficient strategy against uveal melanoma.

Objective:To analyze the effects of prescription reviews of key monitored drugs using the drug regulatory platform in a district of Shanghai, and the influencing factors for the rate of unqualified prescriptions.Methods:A drug regulatory platform in a district in Shanghai began to implement prescription reviews since June 2018, extracting the reviewed prescriptions of all key monitored drugs and related information from June to December. Taking the unqualified rate of prescription in June as the baseline data, the χ2 test and the logistics regression model were used for data analysis. Results:From June to December 2018, clinical pharmacists reviewed 19 084 outpatient and emergency prescriptions and 10 607 inpatient prescriptions. The unqualified rates of outpatient and emergency prescriptions and inpatient prescriptions dropped from 16.52% and 23.28% in June to 4.45% and 13.80% in December, respectively. Logistics regression analysis showed that the rate of unqualified reviews of outpatient and emergency prescriptions was lower than that of inpatient prescriptions, and the rate of unqualified prescriptions in hospital was significantly lower than that in community health service centers; the rate of unqualified prescriptions for auxiliary drugs and nutritional drugs was significantly lower than that of antibacterial drugs.Conclusions:The prescription reviews of key monitored drugs can effectively promote the rational use of drugs. In the future, it is recommended to strengthen technical support for community health service centers and professional training for clinicians and pharmacists.

Seventy two patients with initial cerebral infarction admitted in the Second Hospital of Jiaxing from March 2017 to October 2017 were enrolled. Patients underwent conventional ultrasonography and contrast-enhanced ultrasonography for two-dimensional echo grading and neovascularization grading of carotid artery plaques respectively. There were 113 carotid plaques in 72 patients with cerebral infarction,27 cases recurred after 1 years with 44 patches. The echo grading of recurrence group was mainly grade Ⅰ and grade Ⅱ,the number of Ⅰ,Ⅱ,and Ⅲ plaques was 23,18,and 3,respectively. The echo grading of non-recurrence group was mainly grade Ⅲ,the number of Ⅰ,Ⅱ,and Ⅲ plaques was 36,23,and 40,respectively. The echo grading of carotid artery plaque between the two groups was significantly different (P<0.05). The grade of carotid neovascularization in recurrence group was mainly grade Ⅲ and grade Ⅳ,the number of Ⅰ,Ⅱ,Ⅲ and Ⅳ plaques was 2,5,27 and 10,respectively. The echo grading of non-recurrence group was mainly grade Ⅰ and grade Ⅱ,the number of Ⅰ,Ⅱ,Ⅲ and Ⅳ plaques was 24,52,14 and 9,respectively. There was significant difference in the grade of carotid neovascularization between the two groups (P<0.05). The area under curve(AUC) of echo grade<Ⅱ,echo grade<Ⅲ,neovascularization grade>Ⅰ,neovascularization grade>Ⅱ and neovascularization grade>Ⅲ in predicting the recurrence of cerebral infarction were 0.553,0.641,0.587,0.793 and 0.557,respectively,the AUC of neovascularization grade>Ⅱ was significantly higher than the other four criteria (P<0.01). The recurrence curves of carotid plaques with different neovascularization grades were significantly different (χ²=49.18,P<0.01). Carotid plaque vulnerability evaluated by contrast-enhanced ultrasonography is an effective indicator for predicting recurrence of cerebral infarction.

Objective To explore the effectiveness of endoscopic ultrasound( EUS)-guided holmium laser ablation for primary pancreatic implantation tumor in nude mice. Methods Pancreatic cancer cell line SW1990 were implanted into 204-6-week-old male balb/c nude mice to establish primary pancreatic implantation tumor in situ models. Then the nude mice were randomly divided into two groups, the treatment group(n=10) and the control group(n=10). The treatment group underwent EUS-guided holmium laser ablation in the pancreatic tumor. And no interventions were given to the control group. The volume of tumors of the two groups were measured under EUS at time points of 7 d, 14 d and 28 d after ablation. The activities, appetites and psychosis of all nude mice were evaluated in the meantime. At 28 d after ablation, lesions of pancreas were dissected and sliced for H&E staining. Results There were no complications in the treatment group, and all nude mice could tolerate the procedure. The mental state, activities and appetites of nude mice in the experimental group were better than those in the control group. Tumors of the control group enlarged. There was significant difference in the tumor size between the two groups at 28 d after ablation. HE staining showed coagulation necrosis in the ablation area. Conclusion EUS-guided holmium laser, producing coagulative necrosis in the ablation area, is effective for primary pancreatic implantation carcinoma in nude mice for about 28 days.

Objective To analyze set-up errors for irradiation of nasopharyngeal carcinoma by using kilo-voltage cone beam computed tomography (kV-KBCT) scanning, and to calculate the external margin from planning target volume (PTV) of nasopharyngeal carcinoma. Methods A total of 150 nasopharyngeal carcinoma patients in Meizhou People's Hospital from December 2014 to November 2016 were treated by image-guided radiation therapy (IGRT), kV-KBCT, CT image scanning matcthed by bone and grey alignment. PTV of nasopharyngeal carcinoma was also calculated. Results According to PTV formula, the external distance before radiotherapy guided by grey alignment was 0.5 mm in X-axis, 0.4 mm in Y-axis, 0.8 mm in Z-axis. While the distance was 0.1 mm in X-axis, 0.5 mm in Y-axis, 1.7 mm in Z-axis guided by bone alignment. After radiotherapy, the external distance guided by grey alignment was 0.4 mm in X-axis, 0.5 mm in Y-axis, 0.9 mm in Z-axis. While the distance was 0.1 mm in X-axis, 0.9 mm in Y-axis, 2.0 mm in Z-axis guided by bone alignment. There was no significant difference in set-up errors of 3 directions and 2 aligned ways before and after treatment.Conclusions The PTV within 3 mm is safe when IGRT is used for directing radiotherapy of nasopharyngeal carcinoma,and kV-KBCT is an effective image equipment.