BACKGROUND:Chitosan modification for magnetic iron oxide nanoparticles can improve the agglomeration and stability of magnetic iron oxide nanoparticles, and in addition, it can be used for tumor hyperthermia and gene therapy in the future.
OBJECTIVE:To explore the biocompatibility of magnetic chitosan microspheres which have the potential application in tumor hyperthermia and gene therapy.
METHODS:Magnetic iron oxide nanoparticles were prepared using the modified chemical precipitation. Phacoemulsification method was used to add chitosan into magnetic iron oxide nanoparticles in the preparation of magnetic chitosan microspheres. Then, the fol owing tests were performed. (1) Cytotoxicity test:L-929 cells were cultured in 1640 medium, polyacrylamide monomer solution, 100%, 75%, 50%, 25%of magnetic chitosan microsphere extracts. (2) Hemolysis test:Magnetic chitosan microspheres leaching solution, saline and distil ed water were added. (3) Micronucleus test:Kunming mice were intraperitoneal y injected with magnetic chitosan microspheres suspension containing 5, 3.75, 2.5, 1.25 g/kg iron oxide magnetic fluid, cyclophosphamide and saline, respectively.
RESULTS AND CONCLUSION:Magnetic chitosan microspheres of 200-300 nm in diameter had an increased dispersion effect. The result of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that the toxicity of the material on L-929 celllines belonged to no cytotoxicity. In the hemolysis test, the hemolysis rate of magnetic chitosan microspheres was 0.69%far less than 5%. In the micronucleus test, magnetic chitosan microspheres suspension did not cause DNA fragmentation and aneuploidy, and did not result in genetic toxicological effects produced by micronuclei. The magnetic chitosan microspheres did not appear with cacogenesis and mutagenesis. From the results, magnetic chitosan microspheres are a kind of high biocompatibility material.

BACKGROUND: Oxygen-glucose depdvation (OGD) in cultured neurons simulates stroke to a certain degree and plays an important role in studying processing and pathophysiological mechanism of ischemic neuronal injury.OBJECTIVE: To produce experimental OGD models in cultured neurons.DESIGN, TIME AND SETTING: A grouping controlled study was performed at the Center Laboratory of Third Hospital, Peking University from January 2007 to March 2008.MATERIALS: Fetal Wistar rats with gestational age of 17-19 days were collected in this study.METHODS: Primary cultures of cortical neurons that were derived from fetal Wistar rats with gestational age of 17-19 days were performed to remove pollutional non-neuronal cells. OGD was produced by incubation with non-glucose balanced salt solution and 2% Oxyrasa in 7-day cultured cortical neuron cultures. Cell cultures were kept in a humidified 37 ℃ incubator. In the control group, cell culture medium was replaced with balanced salt solution containing 20 mmol/L glucose. In the sham operation group,balanced salt solution containing 20 mmol/L glucose and Oxyrasawere used to replace the medium.MAIN OUTCOME MEASURES: Oxygen concentration in the culture medium was measured with blood gas analysis; neuronal death in the experimental group was observed under phase contrast microscope; lactate dehydrogenase activity was detected with lactate dehydrogenasa assay; effect of oxygen-glucose deprivation on neuronal viability was observed with trypan blue staining.RESULTS: Measurement of oxygen concentration showed that hypoxia could be quickly achieved shortly after the addition of Oxyrase; lactate dehydrogenase assay revealed that after treatments of neuron cultures with Oxyrase and non-glucose balanced salt solution, lactate dehydrogenase release increased significantly with the treatment time; trypan blue staining and phase contrast microscope showed that cell viability decreased after treatments of Oxyrase and non-glucose balanced salt solution, and most neurons died 6 hours after OGD.CONCLUSION: These results show that Oxyrase, together with non-glucose balanced salt solution, can be conveniently used to produce OGD condition in cultured neuronal cells which is greatly useful in the study of simulating cerebral ischemia in vitro.

Objective To obtain purified ClpP produced by prokaryotic expression system,and to evaluate the protection effect elicited by ClpP in animal protection test.Methods The template DNA was isolated from the culture of TIGR4 Streptococcus pneumoniae.The complete ClpP open reading frame(ORF)was cloned into pET-32a expression vector by gene recombination technology in vitro.After prokaryotic expression,purification and sequence identification,the recombinant ClpP were innoculated into mice,and at the same time a group of mice were inoculated with the antibody to ClpP.We monitored the survival time of the innoculated mice after being challenged intraperitoneally with,TIGR4.Results We obtained high-expressed recombinant antigen protein which was then identified by Western blot,and we have got the recombinant antigen protein with a purity of more than 90%after being purified through Ni2+ affinity chromatography and processed by dialysis.The sunrvival time of mice immunized with ClpP or ClpP antibody were significantly langer than that of the mice received PBS.Conclusion The recombinant ClpP antigen protein can elicit protection to the invasive S.pneumoniae infection in mice,which might make ClpP as a candidate of S.pneumoniae vaccine.

AIM: To investigate the neuroprotective effect of tubuloside B,one of the phenylethanoids isolated from the stems of Cistanche salsa,on H2O2-induced injury in PC12 cells.METHODS: PC12 cells were exposed to various doses of tubuloside B for 12 h,then treated with H2O2 at concentration of 100 ?mol/L for 24 h.The cell viability was observed with MTT assay.Reactive oxygen species and the mitochondrial membrane potential were measured with laser scanning confocal microscopy(LSCM).The DNA content and percentage of apoptosis were assayed by DNA agarose gel electrophoresis and flow cytometry.The activation of caspase-3 was detected with the caspase-3 activity assay kit.RESULTS: Following treatment with H2O2 for 24 h,H2O2 induced a significant decrease in cell viability;DNA ladder was observed and apoptosis percentage was as high as 48.0%.Accumulation of intracellular ROS,increase in caspase-3 activity and the decrease in mitochondrial membrane potential as indicated with the decrease of red/green ratios(from 5.97 to 0.41) were detected.However,pretreatment with tubuloside B(1,10 or 100 mg?L-1) for 12 h exhibited cytoprotective effects in a dose-dependent manner.Tubuloside B obviously enhanced the cell viability,reduced formation of the DNA ladder,and significantly reduced the number of cells labeled with Annexin-V.The percentage of apoptosis/necrosis neurons was significantly decreased to 30.9%,18.3% and 6.2%,respectively.LSCM showed that the tubuloside B attenuated the accumulation of ROS and the H2O2-induced collapse of mitochondrial membrane potential in PC12 cells.The significant decrease in caspase-3 activity was detected,compared to the H2O2-treated cells at the same time point.CONCLUSION: Tubuloside B has the neuroprotective capacity to antagonize H2O2-induced apoptosis and injury in PC12 cells,indicating it may be useful for treating some neurodegenerative diseases.

Aim To investigate the neuroprotective effect of verbascoside,one of the phenylethanoids isolated from the stems of Cistanche salsa,on MPP+-induced injury in SHSY5Y cells.Methods SHSY5Y cells were exposed to various doses of verbascoside for 12 h,and then treated with 200 ?mol?L-1 MPP+ for 24 h.The cell viability was observed with MTT assay;reactive oxygen species,the mitochondrial membrane potential and the percentage of apoptosis were measured by flowcytometer;the activation of caspase-3 was measured with the caspase-3 activity assay kit;the expression of Bcl-2 was measured with Western blot.Results Following treatment with MPP+ for 24 h,MPP+ induced a significant decrease of cell viability;apoptosis percentage were 38.9%;accumulation of intracellular ROS,increase of caspase-3 activity and the decreases in mitochondrial membrane potential were detected.However,pretreatment with verbascoside (0.1,1 or 10 mg?L-1) for 12 h exhibited cytoprotective effects in a dose-dependent manner.Verbascoside obviously enhanced cell viability,and significantly reduced the number of cells labeled with Annexin-V.The percentage of apoptosis neurons was significantly decreased to 29.5%,15.3% and 8.6% respectively.Flowcytometer showed the verbascoside attenuated the accumulation of ROS and the MPP+-induced collapse of mitochondrial membrane potential in SHSY5Y cells.And significant decreases were detected in caspase-3 activity compared with the MPP+-treated cells at the same time point.Moreover,pretreatment with verbascoside promoted the expression of Bcl-2.Conclusions verbascoside had the neuroprotective capacity to antagonize MPP+-induced apoptosis in SHSY5Y cells,and might be useful in treating Parkinson disease.

Objective　To investigate the prognosis and influencing factors of intravascular interventional therapy for posterior circulation infarction. Method　For selecting our department admitted in October 2019 and October 2017-two years hospitalized with a total of 42 cases of clinically diagnosed patients with posterior circulation infarction，patient gender，age，on admission，the main symptoms and signs，pathogenic factors，pathogenesis time interval，when to see a doctor to vascular disease opening time，lesion blood vessel parts，collateral compensatory，operation method choice and recanalization，vascular interventional treatment and postoperative 7 days before the NIHSS score，GCS score，mRS score when discharged from hospital，postoperative complications and prognosis of 90 days. According to the improved Rankin score of patients with 90-day prognosis，the patients were divided into a good prognosis group （mRS score <4 points） and a poor prognosis group （mRS score ≥ 4 points），and statistical analysis was conducted. Results　（1） For posterior circulation infarction，multiple interventional therapies were used to treat the lesion vessel opening rate of 71.4%，the 3-month mortality rate of posterior circulation infarction was 28.6%. And the good prognosis rate was 50%. （2）Univariate analysis suggested a statistically significant difference in the prognosis of patients with smoking P<0.01）. （3）Logistic regression analysis showed that after adjusting for cerebrovascular risk factors such as smoking，lower basilar artery occlusion was associated with poor prognosis. （4）Good intraoperative lesion vessels （Ⅱb level or Ⅲ level） and is related to good prognosis. Conclusion　Endovascular interventional therapy is an efficient and rapid method for posterior circulation infarction to open blood vessels. Good recanalization of diseased blood vessels during operation is positively correlated with good prognosis，while smoking and lower basilar artery occlusion are correlated with poor prognosis.

The rapid spread of carbapenemase-producing Klebsiella pneumoniae(cpKP)poses seri-ous threats to public health;however,the underlying genetic basis for its dissemination is still unknown.We conducted a comprehensive genomic epidemiology analysis on 420 cpKP isolates col-lected from 70 hospitals in 24 provinces/autonomous regions/municipalities of China during 2009-2017 by short-/long-read sequencing.The results showed that most cpKP isolates were categorized into clonal group 258(CG258),in which ST11 was the dominant clone.Phylogenetic analysis revealed three major clades including the top one of Clade 3 for CG258 cpKP isolates.Additionally,carbapenemase gene analysis indicated that blaKPC was dominant in the cpKP isolates,and most blaKPC genes were located in five major incompatibility(Inc)groups of blaKPC-harboring plasmids.Importantly,three advantageous combinations of host-blaKPC-carrying plasmid(Clade 3.1+3.2-IncFⅡpHN7A8,Clade 3.1+3.2-IncFⅡpHN7A8:IncR,and Clade 3.3-IncFⅡpHN7A8:InCpA1763-KPC)were identified to confer cpKP isolates the advantages in both genotypes(strong correlation/coevolution)and phenotypes(resistance/growth/competition)to facilitate the nationwide spread of ST11/CG258 cpKP.Intriguingly,Bayesian skyline analysis illustrated that the three advanta-geous combinations might be directly associated with the strong population expansion during 2007-2008 and subsequent maintenance of the population of ST11/CG258 cpKP after 2008.We then examined drug resistance profiles of these cpKP isolates and proposed combination treatment regimens for CG258/non-CG258 cpKP infections.Thus,the findings of our systematical analysis shed light on the molecular epidemiology and genetic basis for the dissemination of ST11/CG258 cpKP in China,and much emphasis should be given to the close monitoring of advantageous cpKP-plasmid combinations.

Objective:To construct a remote screening network for breast cancer based on automated breast ultrasound (ABUS) and explore the value of ABUS with remote reading for breast cancer screening.Methods:We constructed a remote breast cancer screening network including one remote reading center and 48 image-acquisition centers. We recruited women to participate in breast cancer screening at one of these image-acquisition centers from January 2021 to January 2023. The technicians collected the whole breast images using the ABUS. The images were then sent to the reading center through the PVBUS System and interpreted independently by two radiologists using the Breast Imaging Reporting and Data System (BI-RADS). BI-RADS categories 1 and 2 indicate negative screening results, and women diagnosed with these categories were recommended for annual breast ultrasound screening. BI-RADS categories 3, 4, and 5 indicate positive results. Women with BI-RADS category 3 lesions were recommended for follow-up examinations every 6 months using ABUS or handheld ultrasound, while those with BI-RADS 4 and 5 lesions were suggested to undergo pathological examinations.Results:In our study, we enrolled 10 344 women who completed the ABUS screening and were followed up for more than 12 months. After remote reading, 6 164 women were diagnosed with BI-RADS category 1 and 2 626 woman were within BI-RADS category 2. In contrast, 1 404 women were within BI-RADS category 3, a total of 135 women were within BI-RADS category 4, and 15 women were within BI-RADS category 5. The positive screening rate of ABUS was 15.0% (1 554/10 344). The ABUS with remote reading had a detection rate of 3.7/1 000 (38/10 344) for breast cancer screening, with a sensitivity of 97.4% (38/39) and a specificity of 85.3% (8 789/10 305). Among the 38 breast cancer cases detected, 92.1% (35/38) were invasive carcinomas, and 63.2% (24/38) were stage 0 or Ⅰ breast cancers.Conclusions:Breast cancer screening based on ABUS with remote reading provided an efficient and feasible solution to the problem of unevenly distributed medical resources and medical staff levels in various regions of China, enabling the decentralization of high-quality medical resources and improving the accessibility of high-quality screening services. It has provided an alternative for breast cancer screening in China.

Objective:To construct a remote screening network for breast cancer based on automated breast ultrasound (ABUS) and explore the value of ABUS with remote reading for breast cancer screening.Methods:We constructed a remote breast cancer screening network including one remote reading center and 48 image-acquisition centers. We recruited women to participate in breast cancer screening at one of these image-acquisition centers from January 2021 to January 2023. The technicians collected the whole breast images using the ABUS. The images were then sent to the reading center through the PVBUS System and interpreted independently by two radiologists using the Breast Imaging Reporting and Data System (BI-RADS). BI-RADS categories 1 and 2 indicate negative screening results, and women diagnosed with these categories were recommended for annual breast ultrasound screening. BI-RADS categories 3, 4, and 5 indicate positive results. Women with BI-RADS category 3 lesions were recommended for follow-up examinations every 6 months using ABUS or handheld ultrasound, while those with BI-RADS 4 and 5 lesions were suggested to undergo pathological examinations.Results:In our study, we enrolled 10 344 women who completed the ABUS screening and were followed up for more than 12 months. After remote reading, 6 164 women were diagnosed with BI-RADS category 1 and 2 626 woman were within BI-RADS category 2. In contrast, 1 404 women were within BI-RADS category 3, a total of 135 women were within BI-RADS category 4, and 15 women were within BI-RADS category 5. The positive screening rate of ABUS was 15.0% (1 554/10 344). The ABUS with remote reading had a detection rate of 3.7/1 000 (38/10 344) for breast cancer screening, with a sensitivity of 97.4% (38/39) and a specificity of 85.3% (8 789/10 305). Among the 38 breast cancer cases detected, 92.1% (35/38) were invasive carcinomas, and 63.2% (24/38) were stage 0 or Ⅰ breast cancers.Conclusions:Breast cancer screening based on ABUS with remote reading provided an efficient and feasible solution to the problem of unevenly distributed medical resources and medical staff levels in various regions of China, enabling the decentralization of high-quality medical resources and improving the accessibility of high-quality screening services. It has provided an alternative for breast cancer screening in China.

Objective:Integrating genes and GEO database related cuproptosis chips,to analyze connection between cupropto-sis genes,immune infiltration and myocardial infarction(MI),construct risk prediction model,predict Western medicine and Chi-nese medicine,analyze miRNA-mRNA regulatory network,and to provides a new research direction for the future study of MI-related cuproptosis mechanism and immune infiltration.Methods:By GEO database retrieval of MI related chips,standardized processing and MI-related cuproptosis genes screening were performed,and immunoinfiltration analysis and quantification were performed based on the treated gene expression matrix,correlation between immune infiltrating cells and function was analyzed,as well as their differ-ences in MI group and the control group.Cuproptosis genes that most related to MI in immune infiltration were screened out,and the risk model was constructed to analyze the risk probability of cuproptosis genes in MI.The Enrichr website and Coremine Medical data-base were used to predict cuproptosis-related genes in MI in Western medicine and traditional Chinese medicine.Finally,the up-stream mirnas of FDX1 and SLC31A1 were predicted by miRTarBase,Starbase and Targetscan databases,and miRNA-mRNA regula-tory networks were constructed.Results:Correlation of immune infiltration showed that Tfh cells and B cells had the strongest positive correlation(r=0.68),while regulatory T cells and iDC had the strongest negative correlation(r=-0.63);the difference analysis of im-mune infiltration showed that the differences among mast cells,NK cells and Th1 cells in the MI group at the cellular level were the most significant(P<0.001);and the differences in APC co-inhibition and MHCⅠ at the functional level were the most significant(P<0.001).Six genes with the highest correlation between immune cells and immune function were screened out:ATP7A,DLD,FDX1,LIAS,LIPT1 and SLC31A1.Results of the risk model showed that the high levels of FDX1 and SLC31A1 were negatively correlated with the risk prediction of MI.A total of 21 Western medicines and 30 traditional medicines were predicted by database comparison.miRTarBase,Starbase and Targetscan databases predicted 9 upstream miRNAs of cuproptosis-related genes in MI,including has-miR-122-5p.Conclusion:Tfh cells,B cells,para-inflammatory,typeⅠinterferon response and other related immune cells and functions may play important roles in pathogenesis and prognosis of MI.FDX1 and SLC31A1 as the key genes of cuproptosis process,are negatively correlated with MI.A total of 30 kinds of traditional Chinese medicines including Spirulina,sheep liver,Wen ezhu,Pian jianghuang and Yujin may have potential value in treatment of MI.Finally,9 miRNAs including has-miR-122-5p may play an important role in the regulation of cuproptosis in myocardial infarction.