Objective:To discuss the role of angiopoietin-like protein 6(ANGPTL6)in liver fibrosis,and to analyze the improving effect of engineered exosome(Exo)-delivered ANGPTL6 mRNA on liver fibrosis.Methods:A total of 12 C57BL/6 mice were randomly divided into olive oil group(OIL group)(intraperitoneally injected with olive oil)and carbon tetrachloride(CCl4)group(intraperitoneally injected with a mixture of olive oil and CCl?),with 6 mice in each group;another 12 C57BL/6 mice were randomly divided into control group(fed a with methionine-choline sufficient diet)and methionine-choline deficient(MCD)group(fed a with MCD diet),and two kinds of mouse liver fibrosis models were established.Real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting method were used to detect the ANGPTL6 mRNA and protein expression levels in liver tissue of the mice in various groups.A total of 30 mice were randomly divided into olive oil+phosphate buffered saline(PBS)group(OIL+PBS group)(intraperitoneally injected with olive oil twice a week for 8 weeks,then injected with PBS buffer by tail vein twice a week for 6 weeks),CCl4+Exo-green fluorescent protein(GFP)mRNA group(established liver fibrosis model by intraperitoneal injection of CCl4 mixture and were injected by tail vein with engineered Exo loaded with GFP mRNA for 6 weeks),and CCl?+Exo-ANGPTL6 mRNA group(established liver fibrosis model by intraperitoneal injection of CCl4 mixture and were injected by tail vein with engineered Exo loaded with ANGPTL6 mRNA for 6 weeks),with 10 mice in each group.The mice in CCl4+Exo-GFP mRNA group and CCl4+Exo-ANGPTL6 mRNA group were injected with engineered Exo twice a week,20 μg per mouse each time(volume 100 μL).ELISA method was used to detect the serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)activities in the mice in various groups;Masson staining and Sirius red staining were used to observe the collagen deposition in liver tissue of the mice in various groups;immunohistochemistry method was used to detect the α-smooth muscle actin(α-SMA)expression levels in liver tissue of the mice in various groups;RT-qPCR method was used to detect the expression levels of α-SMA,collagen type Ⅰ alpha 1 chain(Col1a1),transforming growth factor β1(TGF-β1),and tissue inhibitor of metalloproteinase 1(TIMP-1)mRNA in liver tissue of the mice in various groups.Results:The bioinformatics analysis results showed that ANGPTL6 expression was significantly down-regulated in activated hepatic stellate cell(aHSC).The ultrasound examination results showed that the liver surface of the mice in OIL group was fine and smooth;compared with OIL group,the liver section of the mice in CCl? group was rough and uneven.The RT-qPCR and Western blotting results showed that compared with OIL group,the ANGPTL6 mRNA and protein expression levels in liver tissue of the mice in CCl? group were significantly decreased(P<0.05).The engineered Exo extracted from the supernatant of HEK293T cells had intact structure and could be largely enriched in the fibrotic liver after tail vein injection,with GFP protein being largely expressed in the liver.The ELISA assay results showed that compared with OIL+PBS group,the ALT and AST activities in CCl4+Exo-GFP mRNA group were significantly increased(P<0.05);compared with CCl4+Exo-ANGPTL6 mRNA group,the serum ALT and AST activities in CCl4+Exo-GFP mRNA group were significantly decreased(P<0.05).The Masson staining and Sirius red staining results showed that compared with OIL+PBS group,the collagen deposition in liver tissue of the mice in CCl?+Exo-GFP mRNA group was significantly increased,and the relative collagen area was increased(P<0.05);compared with CCl4+Exo-GFP mRNA group,the collagen deposition in tissue liver of the mice in CCl?+Exo-ANGPTL6 mRNA group was significantly decreased,and the relative collagen area was decreased(P<0.05).The immunohistochemistry results showed that compared with OIL+PBS group,the α-SMA protein expression level in liver tissue of the mice in CCl?+Exo-GFP mRNA group was significantly increased(P<0.05);compared with CCl4+Exo-GFP mRNA group,the α-SMA protein expression level in liver tissue of the mice in CCl?+Exo-ANGPTL6 mRNA group was significantly decreased(P<0.05).The RT-qPCR results showed that compared with OIL+PBS group,the expression levels of Col1a1,α-SMA,TGF-β1,and TIMP-1 mRNA in liver tissue of the mice in CCl?+Exo-GFP mRNA group were significantly increased(P<0.05);compared with CCl4+Exo-GFP mRNA group,the expression levels of Col1a1,α-SMA,TGF-β1,and TIMP-1 mRNA in liver tissue of the mice in CCl?+Exo-ANGPTL6 mRNA group were significantly decreased(P<0.05).Conclusion:Engineered Exo-delivered ANGPTL6 mRNA injected via the tail vein in the mice is mainly enriched in the liver,and engineered Exo delivery of ANGPTL6 mRNA has an improving effect on liver fibrosis in the mice.

Objective: To investigate the influencing factors for delay in healthcare-seeking, definitive diagnosis and identification in patients with pulmonary tuberculosis (PTB) in Minhang District, Shanghai Municipality, so as to provide the basis for effectively reducing delay in PTB patients. Methods: Data of PTB patients in Minhang District from 2017 to 2022 were collected from the Infectious Disease Reporting Information System of Chinese Disease Prevention and Control Information System. The prevalence rates of delay in healthcare-seeking, definitive diagnosis and identification were analyzed, and factors affecting delay in healthcare-seeking, definitive diagnosis and identification were identified using multivariable logistic regression models. Results: A total of 4 214 PTB patients were reported in Minhang District from 2017 to 2022, including 2 802 males and 1 412 females, with a male-to-female ratio of 1.98∶1. The majority of patients were aged 25 to <45 years (1 664 cases, 39.49%). The prevalence rates of delay in healthcare-seeking, definitive diagnosis and identification were 36.81%, 30.21% and 38.09%, respectively. Delay in healthcare-seeking was associated with the year (2018, OR=0.708; 2019, OR=0.549; 2020, OR=0.670; 2021, OR=0.682), gender (female, OR=1.199), occupation (worker, OR=1.379; housekeeping service/housework/unemployed, OR=1.481), case identification route (symptom-based consultation, OR=11.159), and level of the first-diagnosed hospital (city-level, OR=1.528). Delay in definitive diagnosis was associated with age (45 to <65 years, OR=1.476), occupation (commercial service, OR=0.687; housekeeping service/housework/unemployed, OR=0.672), household registration (non-local, OR=0.820), case identification route (symptom-based consultation, OR=0.616), pathogen test result (negative/not tested, OR=1.903), and the level of the first-diagnosed hospital (city-level, OR=0.311). Delay in identification was associated with the year (2018, OR=0.785; 2019, OR=0.647; 2020, OR=0.790; 2021, OR=0.710), occupation (commercial service, OR=0.687), household registration (non-local, OR=0.848) and level of the first-diagnosed hospital (city-level, OR=0.560) Conclusions Year, gender, occupation, case identification route and level of the first-diagnosed hospital are influencing factors for delay in healthcare-seeking in PTB patients. Age, occupation, household registration, case identification route, pathogen test result and level of the first-diagnosed hospital are influencing factors for delay in definitive diagnosis. Year, occupation, household registration and level of the first-diagnosed hospital are influencing factors for delay in identification.

Objective To investigate the effects of neurotrophic factor Neuritin overexpression on the angiogenic effects of chicken embryonic allantoic membrane (CAM) and human umbilical vein endothelial cells (HUVECs),and to provide a new direction for the treatment of angiogenic diseases. Methods Thirty fresh yellow-skinned breeding eggs were selected to establish a CAM model,which were divided into three groups by randomized numerical table method:positive control group (bFGF),negative control group (NS) and experimental group (Neuritin),with 10 eggs in each group. The positive control group was loaded with 2500 U/mL of bFGF,the experimental group was loaded with 10 μg/mL of Neuritin protein,and the negative control group was loaded with NS. 10 μL loading volume was loaded into each group,and all CAMs were incubated at the same temperature,relative humidity,and time,and the vascular branching,number,and size of the CAMs in each group were recorded after 72 h of incubation. Fresh umbilical cords from healthy pregnant women were selected to produce primary HUVECs,which were divided into three groups:transfected with recombinant plasmid (HUVEC-neu group),transfected with empty vector (HUVEC-3.1 group),and untransfected (HUVEC group). Primary HUVECs in the HUVEC-neu group were transfected with the recombinant plasmid Neuritin,and those in the HUVEC-3.1 group were transfected with the empty vector. HUVEC-3.1 group was transfected with the empty vector plasmid,and HUVEC group was not given any special treatment,and all three groups received the same culture regimen. Western blot was used to detect the protein expression level of Neuritin in HUVEC-3.1 and HUVEC-neu groups. CCK-8 assay,cell scratch assay,Transwell assay,and tube formation assay were used to detect protein expression level of Neuritin in HUVEC-3.1 group and HUVEC-neu group,and HUVEC-neu groups for cell proliferation,migration and tube formation. Results (1) The number of CAM vessel branch points and microvessels in the experimental group was significantly increased compared with that in the negative control group (P<0.01),but there was no statistically significant difference in the number of large and medium-sized vessels between the two groups (P>0.05);(2) Neuritin was successfully overexpressed in HUVECs in the HUVEC-neu group. (3) Compared with the HUVEC-3.1 group,the proliferation vigor of cells in the HUVEC-neu group was decreased (P<0.05),but their migration and tube formation abilities were significantly enhanced (P<0.01). Conclusion Neuritin overexpression promotes angiogenesis and participates in the regulation of neovascularization by affecting cell prolif-eration,migration,and tube formation ability.

Objective To investigate the effects of neurotrophic factor Neuritin overexpression on the angiogenic effects of chicken embryonic allantoic membrane (CAM) and human umbilical vein endothelial cells (HUVECs),and to provide a new direction for the treatment of angiogenic diseases. Methods Thirty fresh yellow-skinned breeding eggs were selected to establish a CAM model,which were divided into three groups by randomized numerical table method:positive control group (bFGF),negative control group (NS) and experimental group (Neuritin),with 10 eggs in each group. The positive control group was loaded with 2500 U/mL of bFGF,the experimental group was loaded with 10 μg/mL of Neuritin protein,and the negative control group was loaded with NS. 10 μL loading volume was loaded into each group,and all CAMs were incubated at the same temperature,relative humidity,and time,and the vascular branching,number,and size of the CAMs in each group were recorded after 72 h of incubation. Fresh umbilical cords from healthy pregnant women were selected to produce primary HUVECs,which were divided into three groups:transfected with recombinant plasmid (HUVEC-neu group),transfected with empty vector (HUVEC-3.1 group),and untransfected (HUVEC group). Primary HUVECs in the HUVEC-neu group were transfected with the recombinant plasmid Neuritin,and those in the HUVEC-3.1 group were transfected with the empty vector. HUVEC-3.1 group was transfected with the empty vector plasmid,and HUVEC group was not given any special treatment,and all three groups received the same culture regimen. Western blot was used to detect the protein expression level of Neuritin in HUVEC-3.1 and HUVEC-neu groups. CCK-8 assay,cell scratch assay,Transwell assay,and tube formation assay were used to detect protein expression level of Neuritin in HUVEC-3.1 group and HUVEC-neu group,and HUVEC-neu groups for cell proliferation,migration and tube formation. Results (1) The number of CAM vessel branch points and microvessels in the experimental group was significantly increased compared with that in the negative control group (P<0.01),but there was no statistically significant difference in the number of large and medium-sized vessels between the two groups (P>0.05);(2) Neuritin was successfully overexpressed in HUVECs in the HUVEC-neu group. (3) Compared with the HUVEC-3.1 group,the proliferation vigor of cells in the HUVEC-neu group was decreased (P<0.05),but their migration and tube formation abilities were significantly enhanced (P<0.01). Conclusion Neuritin overexpression promotes angiogenesis and participates in the regulation of neovascularization by affecting cell prolif-eration,migration,and tube formation ability.

Background/Aims: The shortage of donor liver hinders the development of liver transplantation. This study aimed to clarify the poor outcomes of functionally marginal liver grafts (FMLs) and provide evidence for the improvement of ischemia-free liver transplantation (IFLT) after FML transplantation. Methods: Propensity score matching was used to control for confounding factors. The outcomes of the control group and FML group were compared to demonstrate the negative impact of FMLs on liver transplantation patients. We compared the clinical improvements of the different surgical types. To elucidate the underlying mechanism, we conducted bioinformatic analysis based on transcriptome and single-cell profiles. Results: FMLs had a significantly greater hazard ratio (HR: 1.969, P=0.018) than did other marginal livers. A worse 90-day survival (Mortality: 12.3% vs. 5.0%, P=0.007) was observed in patients who underwent FML transplantation. Patients who received FMLs had a significant improvement in overall survival after IFLT (Mortality: 10.4% vs 31.3%, P=0.006). Pyroptosis and inflammation were inhibited in patients who underwent IFLT. The infiltration of natural killer cells was lower in liver grafts from these patients. Bulk transcriptome profiles revealed a positive relationship between IL-32 and Caspase 1 (R=0.73, P=0.01) and between IL-32 and Gasdermin D (R=0.84, P=0.0012). Conclusions FML is a more important negative prognostic parameter than other marginal liver parameters. IFLT might ameliorate liver injury in FMLs by inhibiting the infiltration of NK cells, consequently leading to the abortion of IL-32, which drives pyroptosis in monocytes and macrophages.

Alzheimer's disease(AD)is an irreversible neurodegenerative disorder,making early screening and diagno-sis pivotal for its effective treatment.The complexity of diagnosing AD,however,poses significant challenges for primary-level screening.As an extension of the central nervous system,the retina exhibits changes closely linked to AD,offering a new pathway for non-invasive early detection of AD.In recent years,deep learning(DL)has achieved notable advance-ments in the medical field,especially in image recognition and analysis.The synergy between DL and retinal imaging has unveiled substantial potential in diagnosing and treating AD.This article reviews the advancements in applying DL to ana-lyze retinal images related to AD,encompassing the diagnosis,prediction of progression,and long-term management of AD,as well as the current shortcomings in clinical practice,providing a reference for further research into the application of DL in retinal imaging for AD.

Objective:To investigate the effects of hyperthermia on the biological behavior of human laryngeal cancer Hep-2 cisplatin-resistant (Hep-2/CDDP) cell line and its possible mechanism.Methods:Hep-2/CDDP cell line was induced by high impact combined with increasing concentration method. Cell count method was used to detect the cell proliferation ability of Hep-2 parental cell group (Hep-2 cells without cisplatin-resistance and the cells were cultured with RPMI 1640 cultured medium without cisplatin), Hep-2/CDDP cell group and Hep-2/CDDP+cisplatin group (using RPMI 1640 cultured medium including 4 mg/L cisplatin). Hep-2/CDDP cell group and Hep-2 parental cell group were treated with cultured medium including 0, 0.004, 0.04, 0.4, 4, 40 mg/L cisplatin, respectively. The sensitivity of Hep-2/CDDP cells to cisplatin, vincristine and 5-fluorouracil was determined by using methyl thiazolyl tetrazolium (MTT) method. The half inhibitory concentration ( IC50) and resistance index (RI) were also calculated. Hep-2/CDDP cell group was divided into 4 subgroups: the cells in the control group were cultured for 24 h at 37 ℃; the cells in hyperthermia group were treated at 43 ℃ for 2 h and then re-cultured at 37 ℃ for 22 h; the cells in cisplatin group were cultured at 37 ℃ for 24 h in cultured medium containing 4 mg/L cisplatin. The cells in hyperthermia combined with cisplatin group were cultured in cultured medium containing 4 mg/L cisplatin, treated at 43 ℃ for 2 h and then re-cultured at 37 ℃ for 22 h. The effects of hyperthermia combined with cisplatin on the proliferation and early apoptosis of Hep-2/CDDP cells were detected by using MTT and flow cytometry. The interaction of hyperthermia combined with cisplatin on the proliferation and early apoptosis of HEP-2/CDDP cells was observed by using factorial analysis. Western blotting was used to detect the effect of hyperthermia combined with cisplatin on the expressions of wild-type p53 and PI3K in Hep-2/CDDP cells. Hep-2/CDDP cells were divided into 4 groups: the control group (Hep-2/CDDP cells were cultured for 24 h at 37 ℃); chemotherapy group was treated with 12 mg/L vincristine or 9 mg/L 5-fluorouracil; in the hyperthermia group, Hep-2/CDDP cells were treated at 43℃ for 2 h and then re-cultured at 37 ℃ for 22 h; in hyperthermia combined with chemotherapy group, the cells were cultured in a medium containing 12 mg/L vincristine or 9 mg/L 5-fluorouracil, treated at 43 ℃ for 2 h and then re-cultured at 37 ℃ for 22 h. MTT method was used to detect the effect of hyperthermia combined with vincristine and 5-fluorouracil on the proliferation of Hep-2/CDDP cells. Results:Hep-2/CDDP cell line was successfully established. There were no significant differences in the number of cells in Hep-2/CDDP cell group, Hep-2 parental cell line group and Hep-2/CDDP + cisplatin cell group at different time points (all P > 0.05), and the doubling time was 43.8, 40.6 and 43.5 h, respectively. The IC50 of Hep-2 parental cell line group and Hep-2/CDDP cell group to cisplatin was 4.771 mg/L and 42.749 mg/L, respectively, and the RI was 8.960. Hyperthermia combined with cisplatin could inhibit the proliferation of Hep-2/CDDP cells ( F = 327.91, P < 0.05) and promote the early apoptosis of Hep-2/CDDP cells ( F = 724.63, P < 0.05). Factorial analysis showed that hyperthermia combined with cisplatin had an interaction effect on the proliferation and early apoptosis of Hep-2/CDDP cells ( F = 185.68, 472.51, all P < 0.05). Western blotting showed that the relative expression levels of wild-type p53 protein and PI3K protein in the control group, hyperthermia group, cisplatin group and hyperthermia combined with cisplatin group were significantly different ( F = 547.76, 404.44, all P < 0.01). Hyperthermia combined with vincristine or 5-fluorouracil could inhibit the proliferation of Hep-2/CDDP cells ( F = 33.06, 34.61, all P < 0.05). Factorial analysis showed that hyperthermia combined with vincristine and 5-fluorouracil had no interaction effect on the proliferation of Hep-2/CDDP cells ( F = 0.64,0.60, all P > 0.05). Conclusions:Hyperthermia may reverse the resistance of Hep-2/CDDP cell line to cisplatin by upregulating wild-type p53 expression and inhibiting the PI3K pathway. Hep-2/CDDP cell line has cross-resistance to vincristine and 5-fluorouracil. Hyperthermia can increase the sensitivity of Hep-2/CDDP cell line to vincristine and 5-fluorouracil.

Objective To investigate the epidemic characteristics of pulmonary tuberculosis (TB) in Minhang District of Shanghai, and to provide a scientific basis for TB prevention and control. Methods Data from active TB patients registered in Minhang District of Shanghai from 2009 to 2019 was collected and analyzed using descriptive epidemiological method. Results From 2009 to 2019 the registration rates of TB patients in Minhang district of Shanghai showed a downward trend, with an average annual registration rate of 32.35/100 000, including 3 611 (41.08%) patients who were registered permanent residents of Shanghai and 5 179 (58.92%) patients who were non-permanent residents. The ratio of men to women was 2.04:1. Workers accounted for more cases (22.77%) than other occupations among patients without registered permanent residence, while retirees accounted for the majority of cases (38.60%) among patients with registered permanent residence. Among the TB patients without registered residence, the highest proportion (45.86%) was in the 20-29 years old group, while the highest proportion in permanent residents was in the 50-59 years old group (19.47%). The peak incidence occurred in May in patients without registered permanent residence, while the peak incidence was in June in patients with registered permanent residence. Conclusion It is necessary to strengthen the prevention and control of TB in people without registered permanent residence of Shanghai, and at the same time, to strengthen the prevention and control of TB in old people and students with registered permanent residence of Shanghai.

Objective To investigate the clinical significance of restrictive foramen ovale ( RFO ) monitored by fetal echocardiography during the middle to late stage of pregnancy . Methods The detection rate ,echocardiographic features and outcome in fetuses with RFO without cardiac malformations from 7319 pregnant women received prenatal echocardiography were retrospectively reviewed and analyzed . Results RFO was found in 40 of 7319 (0 .55％ ) fetuses . The inclusion criteria including a narrow right to left shunt of less than 2 .5 mm in diameter across atrial septum , enlarged right atrium , increased right-to-left ventricular size ratio ,and increased size ratio of main pulmonary artery to aorta were present in 40 fetuses . The direct ultrasound characters of RFO included limited opening of oval valve ( 70％ ) and foramen ovale diameter less than 2 .5 mm (30％ ) . And atrial septal aneurysm ( 62 .5％ ) ,redundant primum atrial septum (57 .5％ ) ,abnormal ductus arteriosus ( 57 .5％ ) might also be present commonly in RFO . As the gestational weeks increased , the size ratio of right-to-left atrium , right-to-left ventricle and the main pulmonary artery to aorta also increased significantly( P =0 .004 , P <0 .001 , P <0 .001) . Among the 40 fetuses with RFO ,21 cases ( 52 .5％ ) gave birth in full term ,8 cases ( 20％ ) which were detected severe tricuspid regurgitation gave birth in early cesarean section ,5 cases ( 12 .5％ ) had induced labor and 6 cases (15％ ) were lost in the follow-up . Of the 29 newborns ,only 1 case died of heart failure ,and the other 28 subjects recovered both from heart structure or cardiac function within four months . Conclusions RFOwithout cardiac malformations presents echocardiographic features characterized by a narrow right to left shunt of less than 2 .5 mm in diameter across atrial septum . Fetal echocardiography can monitor the dynamic change of fetal heart structure and function based on the increase of right heart load and decrease of left heart volume ,which has important clinical significance for assessing fetal intrauterine condition and prognosis .

Objective To investigate the relationship between CYP 2C19 gene polymorphisms and clopidogrel efficacy in coronary heart disease patients after percutaneous coronary intervention (PCI).Methods From January 2016 to December 2017,62 patients with acute coronary syndromes and treated with PCI in Guizhou Aerospace Hospital were recruited, CYP2C19 genotype, ADP －induced platelet aggregation rate and myocardial enzymes and other indicators were detected before operation .The myocardial enzymes were measured 24 hours after PCI.According to different metabolic types,the patients were grouped,the above indicators were compared.Results The CYP2C19*1/*1 was 37.10%,CYP2C19*1/*2 was 35.48%,CYP2C19*1/*3 was 11.29%,CYP2C19*2/*2 was 12.90%,CYP2C19*2/*3 was 3.23% and CYP2C19*3/*3 was 0.00%.The LDH,AST,CK,CK－MB and α－HBDH in the PCI patients after operation were significantly higher than those before operation (t＝0.019,0.040, 0.044,0.022,0.014,all P＜0.05).But ADP induced platelet aggregation rate and myocardial enzymes and other indicators among fast metabolism group,intermediate metabolic group and slow metabolic group had no statistically significant differences (all P ＞0.05).Conclusion CYP2C19 mutation frequency in the Chinese population is relatively large,the sample size of this study is less ,the relationship between clopidogrel resistance and the specific genotype can not be obtained ,it need to increase the sample size and comprehensive multi －factor consideration .