OBJECTIVE To establish a method for simultaneous determination of plasma concentration of lamotrigine（LTG）， levetiracetam（LEV） and perampanel（PER） in children with epilepsy and apply this method in clinical practice. METHODS Plasma proteins were precipitated with acetonitrile. Using PER-D 5 as internal standard， ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS） method was adopted. The determination was performed on ACQUITY UPLC HSS T3 C 18 column with mobile phase consisted of 0.1% formic acid with 5 mmol/L ammonium acetate-acetonitrile （gradient elution） at the flow rate of 0.3 mL/min. The column temperature was 40 ℃， and sample size was 5 μL. The analysis time was 5 min. The electrospray ionization source and multiple reaction monitoring mode were used for positive ion scanning. The ion pairs used for quantitative analysis of LTG， LEV， PER and internal standard were m / z 255.9→144.9， m / z 171.1→126.1， m / z 350.1→219.0 and m / z 354.9→220.2， respectively. The steady-state trough concentrations of the aforementioned drugs in the plasma of 14 pediatric epilepsy patients receiving combination therapy were determined using the same UPLC-MS/MS method as above. RESULTS The linear ranges of LTG， LEV and PER were 0.15-24 μg/mL （ R 2 ＞0.993）， 0.312 5-50 μg/mL （ R 2 ＞0.997） and 6.25-1 000 ng/mL （ R 2 ＞0.997）， respectively. The lower limits of quantification were 0.15 μg/mL， 0.312 5 μg/mL and 6.25 ng/mL， respectively. RSDs of intraday and interday precision tests of the three drugs were no more than 9.83%， and the accuracies （relative errors） were between －9.33% and 13.72%（ n ＝6 or n ＝18）； the average extraction recovery rates were 86.4%-97.9%， and the average matrix effects were 86.9%-110.0% （ n ＝6）. The absolute values of the relative errors in the stability tests were all below 15%. The steady-state trough concentrations of LTG， LEV and PER were （5.64±4.03）μg/mL， （10.67±8.78）μg/mL and（450.20±251.27）ng/mL， respectively； the rates of achieving target trough concentrations were 71.4%， 37.5% and 84.6%， respectively. CONCLUSIONS The established UPLC-MS/MS method is specific， rapid and suitable for the plasma concentration monitoring in epileptic children receiving combination therapy.

Objective This study conducted molecular biological identification of the viruses carried by Culex pipiens pallens specimens collected in Shunyi District, Beijing in 2023, and observed the changes in the infection rate of the viruses carried by Cx. pipiens pallens at different collection times. Methods Cx. pipiens pallens were collected using carbon dioxide mosquito traps. The mosquito samples were ground in batches and analyzed by molecular biology technologies. The virus infection rate at different collection times was analyzed statistically. Results 17 strains of Culex pipiens pallens densovirus(CppDNV)were identified from Cx. pipiens pallens samples collected in Shunyi District, Beijing, in 2023. The nucleotide sequence analysis of the virus genome coding region showed that CppDNV was a single-stranded DNA virus with a total length of 3 335 nt, encoding 2 non-structural proteins(NS1, NS2)and 1 capsid protein(VP). The nucleotide(amino acid)sequence lengths of the three proteins were 2 376 nt(791 aa),1 092 nt(363 aa)and 1 071 nt(356 aa), respectively. Phylogenetic analysis showed that CppDNV was located in genus Brevihamaparvovirus. Statistical analysis showed no significant difference in infection rates across collection times(χ2=4.429, P=0.194). Conclusions CppDNV was identified in Cx. pipiens pallens in Beijing, and it was stably maintained in this natural population.

Objective To investigate the species composition of mosquitoes and breeding conditions of larvae in the rice planting areas of Beijing, and to provide a scientific basis for mosquito control in rice fields. Methods Adult mosquitoes were collected using CO2-baited light traps, and mosquito larvae(including pupae)were sampled via the dipping method. Results A total of 4 genera and 6 species of adult mosquitoes were captured in the study area. The dominant species were Culex pipiens pallens, Cx. tritaeniorhynchus, and Anopheles sinensis. The average adult mosquito density was 5.11 specimens per trap-hour, with slightly lower density in July compared to August. For larvae and pupae,3 genera and 4 species were identified, dominated by Cx. tritaeniorhynchus, An. sinensis, and Aedes vexans. Conclusions The mosquito species composition in Beijing′s rice planting areas is diverse. Mosquito control strategies should prioritize the management of adult mosquitoes, while density control of mosquito larvae in paddy fields can be achieved through intermittent irrigation techniques.

Objective To investigate the causes and management strategies for lower limb ischemic necrosis following xenogeneic heterotopic heart transplantation from a multigene-edited pig to a rhesus monkey. Methods A xenogeneic heterotopic heart transplantation was performed on December 16, 2023, at the Institute of Experimental Animals of Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, using a quintuple-gene-edited pig as the donor and a rhesus monkey as the recipient. On postoperative day (POD) 9, the recipient monkey underwent left lower limb amputation due to ischemic necrosis. Blood samples were collected at various time points after transplantation for analysis of hematologic parameters, liver and renal function, myocardial enzymes, and coagulation profiles. Ultrasound and computed tomography (CT) were used to evaluate anastomotic patency and cardiac structure. Immunological assays, including complement-dependent cytotoxicity (CDC) and IgG/IgM antibody detection, combined with clinical observations, were employed to assess rejection type and therapeutic response. Results The recipient monkey survived for 46 days after transplantation. Echocardiography demonstrated preserved biventricular systolic function in the recipient’s native heart, with left ventricular ejection fraction (LVEF) consistently exceeding 50%. In the donor pig heart, left ventricular endocardial thickening was noted on POD 9, followed by right ventricular endocardial thickening on POD 24, while LVEF remained around 35%. No hyperacute or acute rejection was detected immunologically. CDC positivity ranged between 3.4% and 5.1%, with IgG/IgM antibody binding trends consistent with CDC results. Following amputation, the recipient exhibited elevated inflammatory markers, coagulopathy, and reactive thrombocytosis, which later normalized. Immunohistochemical staining of the necrotic limb revealed arterial and venous thrombosis; however, no T-cell or B-cell infiltration was observed in vascular structures, thrombi, nerves, muscles, fascia, or skin tissues, with CD3 and CD20 staining both negative. Conclusion Limb ischemia after xenogeneic heart transplantation may be associated with lower extremity vascular thrombosis triggered by local trauma in the context of transplantation-induced inflammatory activation and coagulation dysfunction. While no clear lymphocyte-mediated rejection was observed, further studies are needed to explore the potential role of non-lymphocyte-mediated immune mechanisms.

The molecular mechanism of verbascoside(OC1) in promoting acetylcholine(ACh) release in the pathogenesis of Alzheimer's disease(AD) was studied. Adrenal pheochromocytoma cells(PC12) of rats induced by β-amyloid protein(1-42)(Aβ_(1-42)) were used as AD models in vitro and were divided into control group, model group(Aβ_(1-42) 10 μmol·L~(-1)), OC1 treatment group(2 and 10 μg·mL~(-1)). The effect of OC1 on phosphorylated proteins in AD models was analyzed by whole protein phosphorylation quantitative omics, and the selectivity of OC1 for calcium channel subtypes was virtually screened in combination with computer-aided drug design. The fluorescence probe Fluo-3/AM was used to detect Ca~(2+) concentration in cells. Western blot analysis was performed to detect the effects of OC1 on the expression of phosphorylated calmodulin-dependent protein kinase Ⅱ(p-CaMKⅡ, Thr286) and synaptic vesicle-related proteins, and UPLC/Q Exactive MS was used to detect the effects of OC1 on ACh release in AD models. The effects of OC1 on acetylcholine esterase(AChE) activity in AD models were detected. The results showed that the differentially modified proteins in the model group and the OC1 treatment group were related to calcium channel activation at three levels: GO classification, KEGG pathway, and protein domain. The results of molecular docking revealed the dominant role of L-type calcium channels. Fluo-3/AM fluorescence intensity decreased under the presence of Ca~(2+) chelating agent ethylene glycol tetraacetic acid(EGTA), L-type calcium channel blocker verapamil, and N-type calcium channel blocker conotoxin, and the effect of verapamil was stronger than that of conotoxin. This confirmed that OC1 promoted extracellular Ca~(2+) influx mainly through its interaction with L-type calcium channel protein. In addition, proteomic analysis and Western blot results showed that the expression of p-CaMKⅡ and downstream vesicle-related proteins was up-regulated after OC1 treatment, indicating that OC1 acted on vesicle-related proteins by activating CaMKⅡ and participated in synaptic remodeling and transmitter release, thus affecting learning and memory. OC1 also decreased the activity of AChE and prolonged the action time of ACh in synaptic gaps.
Animals ; Rats ; Glucosides/administration & dosage* ; Acetylcholine/metabolism* ; Alzheimer Disease/genetics* ; PC12 Cells ; Phenols/chemistry* ; Neurotransmitter Agents/metabolism* ; Drugs, Chinese Herbal ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics* ; Humans ; Phosphorylation/drug effects* ; Calcium/metabolism* ; Polyphenols

Anxiety disorder is a highly prevalent psychological illness, and research has shown that obesity is a significant risk factor for its development. This study explored the ameliorative effects and mechanisms of saponins from Panax japonicus(SPJ) on anxiety disorder in mice fed a high-fat diet(HFD). Fifty C57BL/6J mice were randomly divided into normal control diet(NCD) group, HFD group, and low-and high-dose SPJ groups. At week 12, six mice from the HFD group were further divided into a control group(treated with DMSO) and an exogenous fibroblast growth factor 21(FGF21) group(administered rFGF21). The anxiety-like behavior of the mice was assessed using the open field test and elevated plus maze test. Hematoxylin-eosin(HE) staining and oil red O staining were performed to observe pathological changes in the liver and adipose tissue. Glucose metabolism was evaluated through the glucose tolerance test(GTT) and insulin tolerance test(ITT). Western blot analysis was performed to detect the expression of FGF21 and its downstream-related proteins in the liver and cortex, along with the expression of brain-derived neurotrophic factor(BDNF), disks large homolog 4(DLG4), and synaptophysin(SYP) in the cortex. Real-time quantitative fluorescent PCR(qPCR) was used to detect the expression of FGF21 and its receptor genes in the liver and cortex. Immunofluorescence staining was employed to examine the expression of neuronal activator c-Fos, FGF21, and the FGF21 co-receptor β-klotho in the cerebral cortex. The results showed that SPJ significantly improved the frequency of activity in the open arms of the elevated plus maze and the central area of the open field in HFD mice, up-regulated the expression of BDNF, DLG4, and SYP, and effectively alleviated anxiety-like behaviors in HFD mice. Compared with the NCD group, HFD mice exhibited up-regulated expression of FGF21 in the liver and cerebral cortex, while the expression of fibroblast growth factor receptor 1(FGFR1) and β-klotho was significantly down-regulated, suggesting that HFD mice exhibited FGF21 resistance. SPJ markedly up-regulated the β-klotho levels in HFD mice, reversing FGF21 resistance. Further comparison with exogenously administered FGF21 revealed that SPJ activates brain cortical regions in a consistent manner, and additionally, SPJ promotes the number and colocalization of c-Fos and β-klotho positive cells in the brain cortex. In summary, SPJ effectively alleviates anxiety-like behaviors in HFD mice. Its mechanism is associated with up-regulation of β-klotho expression in the brain, reversal of FGF21 resistance, and subsequent activation of neurons in the cerebral cortex and amygdala.
Animals ; Diet, High-Fat/adverse effects* ; Fibroblast Growth Factors/genetics* ; Mice ; Male ; Panax/chemistry* ; Mice, Inbred C57BL ; Anxiety/etiology* ; Saponins/administration & dosage* ; Brain-Derived Neurotrophic Factor/genetics* ; Humans ; Liver/metabolism* ; Drugs, Chinese Herbal/administration & dosage*

This study aims to comprehensively analyze the material basis of toad visceral oil(hereafter referred to as toad oil), and explore the pharmacological effect of toad oil on atopic dermatitis(AD). Ultra-high performance liquid chromatography-linear ion trap/orbitrap high-resolution mass spectrometry(UHPLC-LTQ-Orbitrap-MS) and gas chromatography-mass spectrometry(GC-MS) were employed to comprehensively identify the chemical components in toad oil. The animal model of AD was prepared by the hapten stimulation method. The modeled animals were respectively administrated with positive drug(0.1% hydrocortisone butyrate cream) and low-and high-doses(1%, 10%) of toad oil by gavage. The effect of toad oil on AD was evaluated with the AD score, ear swelling rate, spleen index, and pathological section results as indicators. A total of 99 components were identified by UHPLC-LTQ-Orbitrap-MS, including 14 bufadienolides, 7 fatty acids, 6 alkaloids, 10 ketones, 18 amides, and other compounds. After methylation of toad oil samples, a total of 20 compounds were identified by GC-MS. Compared with the model group, the low-and high-dose toad oil groups showed declined AD score, ear swelling rate, and spleen index, alleviated skin lesions, and reduced infiltrating mast cells. This study comprehensively analyzes the chemical composition and clarifies the material basis of toad oil. Meanwhile, this study proves that toad oil has a good therapeutic effect on AD and is a reserve resource of traditional Chinese medicine for external use in the treatment of AD.
Animals ; Dermatitis, Atopic/immunology* ; Disease Models, Animal ; Mice ; Male ; Gas Chromatography-Mass Spectrometry ; Humans ; Bufonidae ; Oils/administration & dosage* ; Chromatography, High Pressure Liquid ; Female ; Mice, Inbred BALB C

Hemodynamics numerical simulation,a multidisciplinary research approach integrating fluid dynamics,clinical medicine,and computer simulation techniques,offers an objective and quantitative analysis of cardiovascular blood flow dynamics through calculating data such as flow rate,pressure,resistance,and wall stress.This review provides a comprehensive overview of the modeling methods and characteristics of numerical simulations within the cardiovascular system.Additionally,it also summarizes the research advancements and potential clinical applications of numerical simulations in the context of various cardiovascular diseases,including vascular aneurysms,aortic dissection,atherosclerosis,structural heart diseases,heart failure,ventricular assist devices,cardiogenic shock,and extracorporeal membrane oxygenation.The goal is to facilitate the deep integration of clinical medicine with engineering technologies,thereby fostering innovative solutions for the precise diagnosis and treatment of cardiovascular disease.

Objective To discuss the clinical efficacy of microwave ablation(MWA)combined with percutaneous vertebroplasty(PVP),radiofrequency ablation(RFA)combined with PVP,and simple PVP in the treatment of vertebral metastatic tumors.Methods A total of 65 patients with vertebral metastatic tumors,who were admitted to the Northern Jiangsu People's Hospital of China to receive treatment from January 2019 to June 2023,were enrolled in this study.The patients were divided into MWA plus PVP group(M+P group,n=25,27 diseased vertebral bodies in total),RFA plus PVP group(R+P group,n=20,23 diseased vertebral bodies in total),and simple PVP group(P group,n=20,24 diseased vertebral bodies in total).Visual analog scale(VAS)score was used to assess the preoperative pain degree and the postoperative relief degree.Bone cement distribution and leakage at one week after surgery were evaluated.Results Successful operation was accomplished in all of the patients.No serious procedure-related complications occurred in all the patients of three groups.In R+P group,P group and M+P group,the preoperative mean VAS scores were(8.48±0.80)points,(8.57±0.98)points and(8.20±1.00)points respectively;the differences among the three groups were not statistically significant(P＞0.05).One week after operation,the pain was significantly relieved in all the patients of three groups;the mean VAS scores in R+P group,P group and M+P group were(4.10±0.85)points,(3.17±0.93)points and(2.44±1.23)points respectively,and the reduction in VAS score was most pronounced in M+P group(P＜0.05).Six months after operation;the mean VAS scores in R+P group,P group and M+P group were(1.87±0.84)points,(4.60±1.09)points and(1.48±0.71)points respectively;and the reduction in VAS score was most pronounced in the M+P group(P＜0.05).The used amount of bone cement in M+P group,R+P group and P group was(7.54±1.44)mL,(5.48±1.12)mL and(4.59±1.56)mL respectively,the difference among the three groups was statistically significant(P＜0.05).The vascular leakage rate(34.8％)and non-vascular leakage rate(52.2％)in P group were remarkably higher than those in R+P group and in M+P group(P＜0.05).No statistically significant difference in the rate of cement leakage existed between R+P group and M+P group(P＞0.05).Conclusion For the treatment of vertebral metastases,MW A plus PVP is superior to RFA plus PVP in pain relief rate.

Objective This research was performed to identify rodent-borne pathogens in Hekou Port,Yunnan Province.Methods Rodents were captured using cages and dissected to collect their lungs,liver,spleen,and other viscera.Eight pathogens,including Yersinia pestis,Leptospira,Bartonella,and Anaplasmataceae,were identified using polymerase chain reaction amplification.Amplified pathogen sequences from positive samples were sequenced,and BLAST homology searches were conducted using GenBank to confirm pathogen identities.A phylogenetic tree of the identified pathogens was constructed using the neighbor joining method.Results The total of 31 rodents,identified as Rattus tanezumi,R.norvegicus,and Mus musculus,were captured.Among these,R.tanezumi was the dominant species,accounting for 64.52％of the total.Two pathogens,Leptospira interrogans and Neoehrlichia mikurensis,were detected,with positivity rates of 9.68％and 29.03％,respectively.No other pathogens were detected.The overall positivity rate for rodent-borne pathogens was 35.48％.Conclusions The single 16S rRNA gene fragment is insufficient for the molecular identification of all Neoehrlichia species.Accurate species identification should be based on a combined analysis of multiple genes.The prevalence of rodent-borne pathogens in Hekou Port indicates the necessity for enhanced surveillance of rodent-borne diseases and implementation of additional prevention and control measures in border ports.