ObjectiveTo compare the differences between the ultrafiltration method and the dextran gel filtration method during the purification of Tfn-modified PCL micelles by using purification efficiency and micelle purity as indicators. MethodsCoumarin-6 （C6） was used as a fluorescent probe and was loaded into HOOC-PEG-PCL to form PCL micelles by the film-dispersion method. Tfn was then conjugated to the surface of PCL micelles via an amidation reaction， resulting in two types of micelles： Tfn/PCL_H and Tfn/PCL_L. The pharmaceutical properties of the two types of micelles were characterized. The micelles were then purified through ultrafiltration method and dextran gel method respectively， and the efficiency of the two methods， along with the purity of the final micelles， was compared. The density of Tfn on the surface of PCL micelles was also calculated. ResultsThe hydrated diameter of PCL micelles was approximately 73 nm， and the C6 loading efficiency was around 0.046%. The size increased to 134 nm and 158 nm for Tfn/PCL_L and Tfn/PCL_H， respectively. The micelle population was monodisperse. The purification results showed that， for the ultrafiltration method， after two and one rounds of purification， the Tfn/C6 ratio stabilized at 23.6 and 3.4 for Tfn/PCL_H and Tfn/PCL_L， respectively. For the dextran gel filtration method， the Tfn/C6 ratio reached 23.7 for the Tfn/PCL_H group after two rounds of purification. However， for the Tfn/PCL_L group， the Tfn/C6 ratio increased during four rounds of dextran gel purification， and a significant difference （P = 0.042 4） was observed between the first and last filtrations. The density of Tfn in the final micelles were calculated. For the ultrafiltration method， the Tfn density of Tfn/PCL_H and Tfn/PCL_L were 94.9% and 13.8%， respectively. For the dextran gel filtration method， the density of the two micelles were 95.6% and 14.4%， respectively. For Tfn/PCL_L group， the density results revealing a statistically significant difference （P=0.000 2）. ConclusionThe purification efficiency of the two methods is comparable. However， the purity of the final micelles shows a significant difference， with the dextran gel filtration method resulting in higher purity， particularly for the Tfn/PCL_L micelles.

AIM:To investigate the impact of core 1 β3-galactosyltransferase-specific molecular chaperone(COSMC)on the stem cell-like properties of gastric adenocarcinoma cells,and to elucidate its underlying mechanism.METHODS:GEPIA(Gene Expression Profiling Interactive Analysis),TIMER(Tumor IMmune Estimation Resource),Wei Sheng Xin platform,UALCAN(University of Alabama at Birmingham Cancer Data Analysis Portal)and UCSC(Uni-versity of California,Santa Cruz)Xena databases were employed to analyze COSMC expression levels across various carci-noma types.The COSMC expression in gastric cancer tissues and cell lines was evaluated using Western blot analysis.Transfection of small interfering RNA was used to knock down COSMC in MKN-45 cell line.In vitro experiments were con-ducted to explore the relationship between the COSMC expression and the stem cell-like properties of gastric cancer.The qPCR,sphere formation assay,soft agar colony formation assay and CCK-8 assay were used to evaluate the effect of COSMC on the stem cell-like properties of gastric cancer cells.Tn+and Tn-cells were isolated using Vicia villosa lectin(VVL)-conjugated immunomagnetic beads to investigate the influence of Tn structure on the stem cell-like characteristics of gastric cancer cells.Finally,the effect of COSMC knockdown on the mitogen-activated protein kinase(MAPK)signal-ing pathway was evaluated by Western blot analysis.RESULTS:(1)The COSMC exhibited differential expression across pancarcinoma,gastric cancer tissues and gastric cancer cell lines,with elevated expression levels in gastric cancer tissues and gastric cancer cell lines compared with normal tissues and gastric epithelial cell line.(2)Knockdown of COSMC sig-nificantly down-regulated the mRNA levels of stem cell markers NANOG,OCT-4 and CD44 in gastric cancer cells,re-duced the efficiency of soft agar colony formation and sphere formation,and increased the sensitivity of the cells to 5-fluo-rouracil(5-FU).(3)The efficiency of tumor sphere formation and soft agar colony formation in Tn-cells,and the resis-tance to 5-FU were significantly higher than those in Tn+cells.(4)Knockdown of COSMC decreased the protein levels of MAPK signaling pathway-related molecules,including c-Jun N-terminal kinase(JNK),p38 and phosphorylated extracel-lular signal-regulated kinase(p-ERK).CONCLUSION:The COSMC has the potential to enhance the stem cell-like properties of gastric cancer cells,which was likely mediated through the activation of the MAPK signaling pathway.

AIM:To investigate the impact of core 1 β3-galactosyltransferase-specific molecular chaperone(COSMC)on the stem cell-like properties of gastric adenocarcinoma cells,and to elucidate its underlying mechanism.METHODS:GEPIA(Gene Expression Profiling Interactive Analysis),TIMER(Tumor IMmune Estimation Resource),Wei Sheng Xin platform,UALCAN(University of Alabama at Birmingham Cancer Data Analysis Portal)and UCSC(Uni-versity of California,Santa Cruz)Xena databases were employed to analyze COSMC expression levels across various carci-noma types.The COSMC expression in gastric cancer tissues and cell lines was evaluated using Western blot analysis.Transfection of small interfering RNA was used to knock down COSMC in MKN-45 cell line.In vitro experiments were con-ducted to explore the relationship between the COSMC expression and the stem cell-like properties of gastric cancer.The qPCR,sphere formation assay,soft agar colony formation assay and CCK-8 assay were used to evaluate the effect of COSMC on the stem cell-like properties of gastric cancer cells.Tn+and Tn-cells were isolated using Vicia villosa lectin(VVL)-conjugated immunomagnetic beads to investigate the influence of Tn structure on the stem cell-like characteristics of gastric cancer cells.Finally,the effect of COSMC knockdown on the mitogen-activated protein kinase(MAPK)signal-ing pathway was evaluated by Western blot analysis.RESULTS:(1)The COSMC exhibited differential expression across pancarcinoma,gastric cancer tissues and gastric cancer cell lines,with elevated expression levels in gastric cancer tissues and gastric cancer cell lines compared with normal tissues and gastric epithelial cell line.(2)Knockdown of COSMC sig-nificantly down-regulated the mRNA levels of stem cell markers NANOG,OCT-4 and CD44 in gastric cancer cells,re-duced the efficiency of soft agar colony formation and sphere formation,and increased the sensitivity of the cells to 5-fluo-rouracil(5-FU).(3)The efficiency of tumor sphere formation and soft agar colony formation in Tn-cells,and the resis-tance to 5-FU were significantly higher than those in Tn+cells.(4)Knockdown of COSMC decreased the protein levels of MAPK signaling pathway-related molecules,including c-Jun N-terminal kinase(JNK),p38 and phosphorylated extracel-lular signal-regulated kinase(p-ERK).CONCLUSION:The COSMC has the potential to enhance the stem cell-like properties of gastric cancer cells,which was likely mediated through the activation of the MAPK signaling pathway.

Intracranial germ cell tumors (iGCT) is a common cause of delayed puberty (DP).　A retrospective analysis was conducted on the clinical data of two patients with DP caused by iGCT. The disease characteristics of the two patients included DP, primary amenorrhea, growth retardation, short stature, subclinical hypothyroidism, etc. They were diagnosed as DP through imaging and related biochemical tests, and some sex hormone drugs were given on the basis of active treatment of the primary disease. Two patients had varying degrees of development in height, weight and secondary sexual characteristics, and further treatment condition is being tracking by us. Early identification and appropriate clinical intervention are of great significance to the reproductive endocrine function, bone health and long-term health such as terminal height of DP patients.

OBJECTIVE To study the effects of transferrin-targeting peptide T7 （7pep） on intracellular transportation of polyethylene glycol-polycaprolactone （PEG-PCL） micelles in human cervical cancer HeLa cells. METHODS Using coumarin-6 （C6） as fluorescent indicator probe， both coumarin-6 （C6）-loaded PEG-PCL （PEG-PCL-C6） micelles and 7pep-modified PEG- PCL （7pep-PEG-PCL-C6） micelles were prepared by film-dispersion method. The particle size， polydispersity index and appearance morphology were compared between two types of micelles； the real-time uptake of two types of micelles by HeLa cells was compared， and the colocalization of two types of micelles with early endosomes （EE）， endocytic recycling compartments （ERC） and late endosomes （LE） after entry into the cells was observed. RESULTS The particle sizes of PEG-PCL-C6 and 7pep-PEG-PCL- C6 micelles were（75.0±2.3）and（82.0±1.5）nm； the polymer dispersity indexes were 0.17±0.20 and 0.17±0.32， respectively， with a regular spherical appearance. The colocalization results showed that entry speed and amount of 7pep-PEG-PCL-C6 micelles were significantly faster/more than those of PEG-PCL-C6 micelles. 7pep-PEG-PCL-C6 micelles entered EE faster than PEG-PCL-C6 micelles， while PEG-PCL-C6 micelles entered ERC at a faster rate than 7pep-PEG-PCL-C6 micelles， and both PEG-PCL-C6 micelles and 7pep-PEG-PCL-C6 micelles tended to accumulate gradually in LE； Pearson coefficient， signal overlap ratio， and colocalization ratio of 7pep-PEG-PCL-C6 micelles with LE were significantly lower 60 minutes after entering the cell than those 30 minutes after entering the cell （P＜0.05 or P＜0.01）. CONCLUSIONS Targeting 7pep modification can increase the entry speed and amount of PEG-PCL-C6 micelles， and also alter their intracellular transportation behavior.

Intracranial germ cell tumors (iGCT) is a common cause of delayed puberty (DP).　A retrospective analysis was conducted on the clinical data of two patients with DP caused by iGCT. The disease characteristics of the two patients included DP, primary amenorrhea, growth retardation, short stature, subclinical hypothyroidism, etc. They were diagnosed as DP through imaging and related biochemical tests, and some sex hormone drugs were given on the basis of active treatment of the primary disease. Two patients had varying degrees of development in height, weight and secondary sexual characteristics, and further treatment condition is being tracking by us. Early identification and appropriate clinical intervention are of great significance to the reproductive endocrine function, bone health and long-term health such as terminal height of DP patients.

Objective:To evaluate the data quality of Shenzhen Type 1 Diabetes Alliance (SZT1D), and to provide a basis for evaluation and improvement for the continuous improvement of data quality.Methods:From December 2018 to July 2021, 697 first-visit type 1 diabetes (T1DM) patients (including 501 in Shenzhen and 196 out-of-Shenzhen) and 120 re-visited T1DM patients (including 113 in Shenzhen and 7 out-of-Shenzhen) who were registered by SZT1D in collaborative research platform network of China Type 1 Diabetes Alliance (hereinafter referred to as China T1D). The data quality was evaluated from three dimensions: data completion, accuracy and revisit. The data completion degree was evaluated by the overall data completion degree and the key indicator completion degree; the data accuracy was evaluated by the probability of abnormal blood glucose value; the patient′s return visit was evaluated by the return visit rate.Results:The main characteristics of T1DM in SZT1D were young and middle-aged adults [age: (34.4±17.1)years] with thin body [BMI: (19.80±3.52)kg/m 2)], half of male and female patients [proportion of male: 52.4%(365/697)]; the main types of diagnosis were classical T1DM [65.22%(150/230)] and latent autoimmune diabetes in adults(LADA) [26.08%(60/230)], and the fasting blood glucose (FPG) [(10.93±6.98)mmol/L] and glycosylated hemoglobin (HbA 1c) [(10.63±3.01)%] were high. The average completion rate of the overall data of the first diagnosed patients in SZT1D was only 60% [(62.9±31.5)%]: the number of patients with overall data completion ≥80% in SZT1D was only 50.2%(350/697); the number of patients with overall data completion ≥80% in Shenzhen was less than that outside Shenzhen [44.3%(222/501) vs 65.3%(128/196), P<0.001]. The key indicators with better completion rate of first-visit were disease course [76.2%(531/697)], age of onset [75.8%(528/697)], family history of diabetes [74.9%(522/697)], etc., but none of them had a completion rate of more than 80%, and the diabetes self-management behavior assessment questionnaire and scale score were completely missing; the frequency of daily blood glucose monitoring [46.1%(231/501) vs 64.3%(126/196), P<0.001], current insulin regimen [44.3%(222/501) vs 63.3%(124/196), P<0.001], number of diabetic ketoacidosis (DKA) since the onset of the disease [45.7%(229/501) vs 64.8%(127/196), P<0.001] and the number of symptomatic hypoglycemia in the past 1 month [39.3%(197/501) vs 63.8%(125/196), P<0.001] were higher in Shenzhen than those reported outside Shenzhen. In addition, the probability of abnormal FPG and postprandial glucose (PPG) [5.2%(24/466); 3.8%(19/236)] were low. The revisit rate was not high [17.2%(120/697)], and the revisit rate in Shenzhen was higher than that outside Shenzhen [22.6%(113/501) vs 3.6%(7/196), P<0.001]. The first revisit rate was 16.2%(113/697) and the second revisit rate was seriously insufficient [1.0%(7/697)]. Conclusions:The data quality of T1DM patients recorded by SZT1D needs to be further improved. Improving the information interconnection between China-T1D and SZT1D, employing quality control personnel and building a systematic data quality evaluation analysis and feedback mechanism are methods to promote the comprehensive, accurate and efficient input of T1DM data and continuously improve the evaluation methods to improve the overall data quality.

Objective To propose a one-way fluid-structure interaction (FSI) method based on an idealized aortic dissection model, so as to analyze the hemodynamics and wall stress in the false lumen (FL) under the influence of multiple overlapping uncovered stents (MOUS). Methods Upon establishment of the numerical model, the models were divided into two categories according to whether the model involved FL perfused branch artery. The characteristics of hemodynamics and wall stress state in the post-operative scenarios were simulated under different surgical strategies. The wall stress state of the FL before and after thrombosis formation was also compared and analyzed. ResultsThe release process of the stents had little influence on wall stress of the FL. The high velocity and high wall shear stress (WSS) area in the FL could not be reduced by using the MOUS alone. If only the proximal entry tear was blocked with a covered stent-graft, the distal end would maintain a region of high flow rate and high WSS. The combination of covered stent-graft and MOUS would result in a region of low flow rate and low WSS, as well as reduced wall pressure and wall stress in the FL. Compared with the model with FL perfused branch arteries, the model without it was more likely to form a region of low flow rate and low WSS after surgery. However, blood pressure in the FL was relatively higher. The formation of thrombus in the FL could greatly reduce wall stress in the area covered by the thrombus. Conclusions The method proposed in this study can simultaneously investigate hemodynamics and wall stress characteristics of the FL, and provide support for studying mechanical mechanism of FL thrombolysis induced by MOUS and the post-operative aortic expansion.

Objective:To investigate the prevalence and characteristics of mcr genes in clinical isolates of Aeromonas spp. in our hospital, and provide reference for clinical analysis of the prevalence and expression of colistin resistance genes. Methods:Polymerase chain reaction (PCR) was used to detect mcr genes in 183 Aeromonas spp. strains. The minimum inhibitory concentrations (MICs) of colistin and polymyxin against mcr-positive Aeromonas spp. were detected by micro broth dilution method. Broth conjugation and filter mating conjugation were performed. Whole genome sequencing was used to analyze the genetic environment of mcr-3 gene in Aeromonas spp.. A recombinant Escherichia coli ( E. coli) DH5α-pGEM-T: : p mcr-3 strain was constructed to verify the expression of mcr-3 gene. Results:The positive rate of mcr-3 gene in 183 strains of Aeromonas spp. was 2.19% (4/183). No mcr-1 or mcr-2 gene was detected among these isolates. Antimicrobial susceptibility test showed that four mcr-3-carrying Aeromonas hydrophilia ( A. hydrophilia) strains were sensitive to colistin and polymyxin (MIC<2 μg/ml). Conjugation experiments indicated that mcr-3 gene could not be transferred between strains. Whole-genome sequencing analysis suggested that the mcr-3 genes carried by the A. hydrophilia isolates belonged to mcr-3.2 and mcr-3-like variants, and no adjacent transfer element was detected upstream and downstream. The recombinant E. coli DH5α-pGEM-T: : p mcr-3 strain was sensitive to colistin (MIC=2 μg/ml). Conclusions:The clinical isolates of A. hydrophilia in our hospital carried mcr-3 gene, but does not exhibit colistin resistance, and no evidence supported the transfer of mcr-3 gene for the time being.

Objective To evaluate the efficiency of the application of array comparative genomic hybridization (array-CGH) in preimplantation genetic diagnosis or screening (PGD/PGS), and compare the clinical outcomes of different stage embryo biopsy. Methods The outcomes of 381 PGD/PGS cycles referred in the First Affiliated Hospital of Nanjing Medical University from July 2011 to August 2015 were retrospectively analyzed. There were 320 PGD cycles with 156 cleavage-stage-biopsy cycles and 164 trophectoderm-biopsy cycles, 61 PGS cycles with 23 cleavage-stage-biopsy cycles and 38 trophectoderm-biopsy cycles.Chromosomal analysis was performed by array-CGH technology combined with whole genome amplification.Single embryo transfer was performed in all transfer cycles.Live birth rate was calculated as the main clinical outcomes. Results The embryo diagnosis rate of PGD/PGS by array-CGH were 96.9%-99.1%. In PGD biopsy cycles, the live birth rate per embryo transfer cycle and live birth rate per embryo biopsy cycle were 50.0%(58/116) and 37.2%(58/156) in cleavage-stage-biopsy group, 67.5%(85/126) and 51.8%(85/164) in trophectoderm-biopsy group (both P<0.01). In PGS biopsy cycles, the live birth rate per embryo transfer cycle and live birth rate per embryo biopsy cycle were the same as 34.8%(8/23) in cleavage-stage-biopsy group, the same as 42.1%(16/38) in trophectoderm-biopsy group (both P>0.05). Conclusions High diagnosis rate and idea live birth rate are achieved in PGD/PGS cycles based on array-CGH technology.The live birth rate of trophectoderm-biopsy group is significantly higher than that of cleavage-stage-biopsy group in PGD cycles;the efficiency of trophectoderm-biopsy is better.