Objective To rescue a strain of Chikungunya virus(CHIKV)using reverse genetics system.Methods Molecular cloning experiments,including PCR,agarose gel electrophoresis and homologous recombination were employed to generate an infectious cDNA clone(pFK-CHIKV-LN)derived from the isolated Caribbean Chikungunya strain(Caribbean strain,isolate M100,Genbank LN898083.1).The mRNA obtained from vitro transcription were transfected into BHK-21 to rescue the CHIKV.The viral titer was determined by 50%tissue culture infection dose(TCID50),with calculations performed using the Reed-Muench method.Western blot(WB)and real time quantitative polymerase chain reaction(RT-qPCR)were employed to assess the levels of viral protein and mRNA at various time points during infection.Results The full-length cDNA clone of CHIKV was successfully constructed,enabling the rescue of CHIKV progeny.RT-qPCR and WB confirmed the significant increasing expression levels of NS1 mRNA and proteins(NS3,E1)of BHK-21 during the infection process.Conclusion The full-length infectious clone of CHIKV has been constructed successfully,which provides a good tool for subsequent studies on the gene structure,protein function and pathogenic mechanism of CHIKV.

In order to explore the effects of lipopolysaccharide(LPS)on the repair of bovine endo-metrial stromal cells(BESCs)during inflammatory response,BESCs were treated by LPS in this study.Cell apoptosis rate was detected using flow cytometry,cell viability was measured using the CCK-8 assay,cell migration ability was observed using a scratch assay,and the expression of con-nective tissue growth factor(CTGF),transforming growth factor-beta 3(TGF-β3)and vascular endothelial growth factor(VEGF)mRNA was measured using qRT-PCR.Additionally,the expression of key proteins in the PI3K/AKT and Wnt/β-catenin signaling pathways was assessed using Western blot analysis.The results showed that cell viability of BESCs significantly decreased(P＜0.01),cell migration ability decreased(P＜0.05),apoptosis rate of BESCs increased(P＜0.01),CTGF and TGF-β3 mRNA expression levels decreased(P＜0.01),while VEGF mRNA ex-pression increased after treatment with LPS(P＜0.01).The phosphorylation levels of PI3K,AKT and GSK-3β proteins decreased(P＜0.05),as well as the expression levels of c-Myc and Cyclin-D1 proteins also decreased(P＜0.01).These results indicated that LPS can inhibit the proliferation of BESCs and promote cell apoptosis possibly through the inhibition of the PI3K/AKT and Wnt/β-catenin signaling pathways.

In order to explore the effects of lipopolysaccharide(LPS)on the repair of bovine endo-metrial stromal cells(BESCs)during inflammatory response,BESCs were treated by LPS in this study.Cell apoptosis rate was detected using flow cytometry,cell viability was measured using the CCK-8 assay,cell migration ability was observed using a scratch assay,and the expression of con-nective tissue growth factor(CTGF),transforming growth factor-beta 3(TGF-β3)and vascular endothelial growth factor(VEGF)mRNA was measured using qRT-PCR.Additionally,the expression of key proteins in the PI3K/AKT and Wnt/β-catenin signaling pathways was assessed using Western blot analysis.The results showed that cell viability of BESCs significantly decreased(P＜0.01),cell migration ability decreased(P＜0.05),apoptosis rate of BESCs increased(P＜0.01),CTGF and TGF-β3 mRNA expression levels decreased(P＜0.01),while VEGF mRNA ex-pression increased after treatment with LPS(P＜0.01).The phosphorylation levels of PI3K,AKT and GSK-3β proteins decreased(P＜0.05),as well as the expression levels of c-Myc and Cyclin-D1 proteins also decreased(P＜0.01).These results indicated that LPS can inhibit the proliferation of BESCs and promote cell apoptosis possibly through the inhibition of the PI3K/AKT and Wnt/β-catenin signaling pathways.

Objective To rescue a strain of Chikungunya virus(CHIKV)using reverse genetics system.Methods Molecular cloning experiments,including PCR,agarose gel electrophoresis and homologous recombination were employed to generate an infectious cDNA clone(pFK-CHIKV-LN)derived from the isolated Caribbean Chikungunya strain(Caribbean strain,isolate M100,Genbank LN898083.1).The mRNA obtained from vitro transcription were transfected into BHK-21 to rescue the CHIKV.The viral titer was determined by 50%tissue culture infection dose(TCID50),with calculations performed using the Reed-Muench method.Western blot(WB)and real time quantitative polymerase chain reaction(RT-qPCR)were employed to assess the levels of viral protein and mRNA at various time points during infection.Results The full-length cDNA clone of CHIKV was successfully constructed,enabling the rescue of CHIKV progeny.RT-qPCR and WB confirmed the significant increasing expression levels of NS1 mRNA and proteins(NS3,E1)of BHK-21 during the infection process.Conclusion The full-length infectious clone of CHIKV has been constructed successfully,which provides a good tool for subsequent studies on the gene structure,protein function and pathogenic mechanism of CHIKV.

Purpose::Mannitol is one of the first-line drugs for reducing cerebral edema through increasing the extracellular osmotic pressure. However, long-term administration of mannitol in the treatment of cerebral edema triggers damage to neurons and astrocytes. Given that neural stem cell (NSC) is a subpopulation of main regenerative cells in the central nervous system after injury, the effect of mannitol on NSC is still elusive. The present study aims to elucidate the role of mannitol in NSC proliferation.Methods::C57 mice were derived from the animal house of Zunyi Medical University. A total of 15 pregnant mice were employed for the purpose of isolating NSCs in this investigation. Initially, mouse primary NSCs were isolated from the embryonic cortex of mice and subsequently identified through immunofluorescence staining. In order to investigate the impact of mannitol on NSC proliferation, both cell counting kit-8 assays and neurospheres formation assays were conducted. The in vitro effects of mannitol were examined at various doses and time points. In order to elucidate the role of Aquaporin 4 (AQP4) in the suppressive effect of mannitol on NSC proliferation, various assays including reverse transcription polymerase chain reaction, western blotting, and immunocytochemistry were conducted on control and mannitol-treated groups. Additionally, the phosphorylated p38 (p-p38) was examined to explore the potential mechanism underlying the inhibitory effect of mannitol on NSC proliferation. Finally, to further confirm the involvement of the p38 mitogen-activated protein kinase-dependent (MAPK) signaling pathway in the observed inhibition of NSC proliferation by mannitol, SB203580 was employed. All data were analyzed using SPSS 20.0 software (SPSS, Inc., Chicago, IL). The statistical analysis among multiple comparisons was performed using one-way analysis of variance (ANOVA), followed by Turkey's post hoc test in case of the data following a normal distribution using a Shapiro-Wilk normality test. Comparisons between 2 groups were determined using Student's t-test, if the data exhibited a normal distribution using a Shapiro-Wilk normality test. Meanwhile, data were shown as median and interquartile range and analyzed using the Mann-Whitney U test, if the data failed the normality test. A p < 0.05 was considered as significant difference. Results::Primary NSC were isolated from the mice, and the characteristics were identified using immunostaining analysis. Thereafter, the results indicated that mannitol held the capability of inhibiting NSC proliferation in a dose-dependent and time-dependent manner using cell counting kit-8, neurospheres formation, and immunostaining of Nestin and Ki67 assays. During the process of mannitol suppressing NSC proliferation, the expression of AQP4 mRNA and protein was downregulated, while the gene expression of p-p38 was elevated by reverse transcription polymerase chain reaction, immunostaining, and western blotting assays. Subsequently, the administration of SB203580, one of the p38 MAPK signaling pathway inhibitors, partially abrogated this inhibitory effect resulting from mannitol, supporting the fact that the p38 MAPK signaling pathway participated in curbing NSC proliferation induced by mannitol.Conclusions::Mannitol inhibits NSC proliferation through downregulating AQP4, while upregulating the expression of p-p38 MAPK.

Objective To investigate the value of wrist MRI in bone age estimation for male adoles-cents in Shanghai,Zhejiang and Jiangsu.Methods A total of 124 Han male adolescents aged 6.0 to 18.0 years from Shanghai,Zhejiang and Jiangsu were selected as subjects.Their weight and height were measured,and T1WI and T2WI sequences of the wrist were scanned.The distal ends of the ra-dius and ulna,and the first to five metacarpal epiphyses and corresponding metaphyses were selected as observational indexes after MRI images of the wrist were obtained.The development of each index was classified(0-2 grades)by a deputy senior imaging expert,then the maximum width of each in-dex was measured by another deputy senior expert.Height,weight,classification and maximum width of indexes were used as input variables,and age was used as the target variable.Support vector ma-chine,random forest,current reality tree,and linear regression models were established to estimate the bone age,and the model with the highest accuracy was selected.Results The height,weight,classifica-tion of wrist bone epiphysis development,maximum width of each bone metaphysis and epiphysis were all correlated with age(P<0.05).The accuracies of the support vector machine were the highest when the differences between bone age and actual chronological age were within 1.0 and 1.5 years(88.7%and 96.0%,respectively).Conclusion It is feasible to estimate bone age by using MRI images.Quantifying the maximum width of the epiphysis and corresponding metaphysis of bone and combining it with MRI image classification can effectively reduce the estimation error.

Objective To analyze the characteristics of drug resistance in patients with disseminated pulmonary tuberculosis,and provide references for the clinical development of individualized treatment plans.Methods A retrospective analysis was conducted on 71 hospitalized patients with disseminated pulmonary tuberculosis treated at the Beijing Chest Hospital,Capital Medical University from January 2015 to January 2024.The susceptibility of Mycobacterium tuberculosis from these patients to 16 anti-tuberculosis drugs was detected using the microplate method for mycobacterial drug susceptibility testing.The study analyzed the culture results of Mycobacterium tuberculosis,drug susceptibility test results,initial treatment or re-treatment status,drug resistance,and differences in drug resistance types between initial and re-treated patients.Results Among the 71 patients with disseminated pulmonary tuberculosis,there were 51 males(71.8%),and 58 cases(81.7%)of acute disseminated pulmonary tuberculosis,with an overall drug resistance rate to 16 anti-tuberculosis drugs of 38.0%.There was no statistically significant difference in the total drug resistance rate between re-treated patients and those undergoing initial treatment[52.2%(12/23)vs.31.3%(15/48),P=0.089].The top 7 drugs to which patients were resistant were streptomycin(Sm)and isoniazid(INH)with 13 cases each(18.3%),rifapentine(Rft)and isoniazid aminosalicylate(Pa)with 10 cases each(14.1%),rifampicin(RFP),rifabutin(Rfb),and capreomycin(Cm)with 9 cases each(12.7%).The top 6 drugs to which initially treated patients were resistant were Cm with 6 cases(12.5%),Sm with 5 cases(10.4%),Pa with 4 cases(8.3%),INH,clarithromycin(Clr),and p-aminosalicylic acid(PAS)with 3 cases each(6.3%).The top 7 drugs to which re-treated patients were resistant were INH with 10 cases(43.5%),Sm,RFP,and Rft with 8 cases each(34.8%),Rfb with 7 cases(30.4%),Pa and levofloxacin(Lfx)with 6 cases each(26.1%).The overall mono-resistance rate,poly-drug resistance rate,and multidrug-resistant rate to 16 anti-tuberculosis drugs were 9.9%,7.0%,and 11.3%,respectively;all mono-resistance patients were initially treated;there was no statistically significant difference in the poly-drug resistance rate between re-treated and initially treated patients(13.0%vs.4.2%,P=0.591),but the multidrug-resistant rate was significantly higher in re-treated patients(30.4%vs.2.1%,P=0.002).Conclusion Drug resistance in disseminated pulmonary tuberculosis is severe.Clinical physicians can develop personalized anti-tuberculosis treatment plans based on drug susceptibility test results.

Objective In this study, electroacupuncture (EA) was used to analyze the expression changes of related proteins in neuroglobin (NGB), PI3K/AKT and apoptotic pathways in the temporal cortex of bilirubin encephalopathy (BE) rats, so as to investigate the therapeutic effect of EA on BE and the relevant mechanism of NGB in this process. Totally 39 seven-day-old SD rats were divided into Sham, BE model and BE+EA groups. The neonatal BE model was established by injecting bilirubin solution (10 μg UCB/g Weight) into the cerebellomedullary cistern, Sham group was injected with the same amount of normal saline. BE rats were treated with EA at Baihui (GV20) and Quchi (LI11) acupoints with the frequency of 2/15 Hz for 15 min. Treatment was performed 12 h before modeling, followed by treatment every 12 h, in a total of three times. HE, Nissl staining and electron microscopy (TEM) were used to observe the pathological and ultrastructural changes of nerve cells in each group. Results showed that EA treatment reduced the damage of cortical neurons of BE rats and increase the number of Nissl bodies. TEM confirmed that EA treatment could alleviate the degree of mitochondria edema. Immunofluorescence staining was used to detect the expression sites and cell types of NGB. Results showed that NGB was mainly expressed in cortical neurons. Western blotting showed that EA treatment increased the expression of NGB, PI3K (p110 alpha), pAKT (Ser473) (P< 0. 05, P< 0. 05 and P< 0. 01, respectively) and the ratio of apoptosis-related protein Bcl-2/Bax (P < 0. 001), decreased the expression of Cleaved Caspase-3 (P< 0. 05) in the temporal cortex of rats. TUNEL staining showed that EA reduced the number of apoptotic cells (BE group 186. 00±13. 86 vs BE+EA group 78. 67±11. 85, P< 0. 01) . This study confirms that EA can promote the expression of NGB in the temporal cortex of BE rats, then activate the PI3K/AKT pathway to exert its neuroprotective function and inhibit the occurrence of apoptosis. EA may become a potential treatment method for BE.

OBJECTIVE: To investigate relationship between cold pain of knee joint and subchondral bone marrow edema (BME). METHODS: From May 2018 to August 2019, 92 patients with knee osteoarthritis (KOA) associated with cold pain of knee were admitted, all patients were underwent MRI examination. The patients were divided into observation group (47 patients with BME) and control group(45 patients without BME). In observation group, there were 6 males and 41 females aged from 36 to 87 years old with an average of (63.2±12.3) years old. In control group, there were 10 males and 35 females, aged from 48 to 84 years old with an average of (62.7±8.3) years old. All patientswere treated with drugs. The degree of joint degeneration was evaluated by Kellgren-Lawrence (K-L) grading. Degree of cold pain of knee was evaluated by knee cold pain score, and degree of BME was evaluated according to WORMS. The correlation between cold pain of knee and K-L grading and BME was analyzed. RESULTS: Score of cold pain in observation group (15.55±7.68) was higher than that of control group (9.42± 5.50), which had significant difference ( CONCLUSION The cold pain of KOA patients is not related to K-L grading, but corelate with BME grading. The Cold pain of knee was more pronounced in KOA patients with BME, and the severity of BME is often related to degree of cold pain. It seemed to be a tendency:the more serious BME, the heavier coldpain.
Adult ; Aged ; Aged, 80 and over ; Bone Marrow ; Edema ; Female ; Humans ; Knee Joint/diagnostic imaging* ; Magnetic Resonance Imaging ; Male ; Middle Aged ; Osteoarthritis, Knee/diagnostic imaging* ; Pain/etiology*

PURPOSE: This study aimed at exploring the application of trauma time axis management in the treatment of severe trauma patients by using the Medicalsystem trauma system. METHODS: We performed a retrospective cohort study involving patients with severe trauma. Patients who were admitted before the application of the Medicalsystem trauma system were divided into before system group; patients who were admitted after the application of the system were divided into after system group. Comparison was made between the two groups. For normally distributed data, means were reported along with standard deviation, and comparisons were made using the independent samples t test. Categorical data were compared using the Chi-square test. The Mann-Whitney U test was used to compare nonparametric variables. RESULTS: There were 528 patients admitted to the study during the study period. There was no significant statistical difference in the time from the start of trauma team to arrive at the resuscitation room between the two groups. The time from arrival at hospital to endotracheal intubation, to ventilator therapy, to blood transfusion, to completion of CT scan, to completion of closed thoracic drainage, to the start of operation, as well as the length of stay in resuscitation room and hospital were significantly lower after the application of the Medicalsystem trauma system. The mortality was decreased by 8.6% in the after system group compared with that in the before system group, but there was no statistical difference. CONCLUSION The Medicalsystem trauma system can optimize diagnosis and treatment process for trauma patients, and accordingly improve the treatment efficiency and shorten the treatment time. Therefore, the Medicalsystem trauma system deserves further popularization and promotion.