AIM:To investigate the effect of doublecortin-like kinase 1(DCLK1)on the biological properties of gastric cancer stem cells,and to explore its possible mechanism.METHODS:Serum-free suspension culture of gastric cancer stem cells and targeted inhibition of DCLK1 activity in gastric cancer stem cells with DCLK1 inhibitor DCLK1-IN-1 were performed.The expression levels of DCLK1,stemness-related proteins(SOX2 and OCT4),proliferation-related pro-teins(cyclin D1 and c-MYC),drug resistance-related proteins(ABCG2 and TOP2A),epithelial-mesenchymal transition-related proteins(E-cadherin,vimentin and Snail),and PI3K/AKT/mTOR signaling pathway-related proteins in gastric cancer stem cells were examined by Western blot.The effects of DCLK1 on viability and drug resistance of gastric cancer stem cells were determined by CCK-8 assay,and the effects of DCLK1 on self-renewal of gastric cancer stem cells were de-termined by methylcellulose spheroid-forming assay.Wound-healing and Transwell assays were performed to assess the ef-fect of DCLK1 on the migration and invasion of gastric cancer stem cells.RESULTS:The expression levels of DCLK1 and stemness-related proteins SOX2 and OCT4 in gastric cancer stem cells were significantly higher than those in parental cells(P＜0.01).The proliferation,drug resistance,migration and invasion of gastric cancer stem cells in DCLK1 inhibi-tion group were significantly lower than those in Sphere cell group(P＜0.01).The expression levels of proliferation-related proteins(c-MYC and cyclin D1)and drug resistance-related proteins(TOP2A and ABCG2)were down-regulated,the ex-pression of epithelial marker E-cadherin was up-regulated,the expression of mesenchymal markers vimentin and Snail was down-regulated,and the expression levels of PI3K/AKT/mTOR signaling pathway-related proteins and their phosphoryla-tion levels were reduced in DCLK1 inhibition group(P＜0.05).CONCLUSION:DCLK1 is highly expressed in gastric cancer stem cells,which may be involved in the proliferation,drug resistance and invasion of gastric cancer stem cells by regulating PI3K/AKT/mTOR signaling pathway.It suggests that DCLK1 can be used as a potential target for gastric cancer stem cells.

Objective:To investigate the role of anti-Müllerian hormone (AMH) on the ovarian function evaluation in premenopausal breast cancer patients with hormone receptor positive after different courses of ovarian function suppression (OFS) treatment.Methods:This study was a two-way cohort study. The main endpoints of the study were menstrual recovery and hormone changes after OFS treatment. Totally sixty-seven premenopausal breast cancer patients were enrolled, whose estrogen receptor (ER) and progesterone receptor (PR) had been tested positive from May 2015 to May 2019, and who had undergone operations, chemotherapies and OFS treatments for 24-60 months in Fudan University Shanghai Cancer Center. Twelve cases were eliminated because of missing tracking. The enrolled cases were divided into 2 groups according to OFS treatment, group A was treated with goserelin for 24 months ( n=32), and group B, with goserelin for 25-60 months ( n=23). Following the OFS treatment, the time points of menstrual recovery were observed and the serum levels of AMH, follicle-stimulating hormone (FSH) and estrogen (E 2) levels were measured in the two groups. Results:The follow-up period of the two groups was (42.0±12.3) months. All the patients survived their follow-ups, except one who developed recurrence and metastasis in the 25-60 month course of OFS. No significant differences were observed in age, height, weight, body mass index (BMI), menarche age, pathological type, pathological grade, ER, PR positive rate, human epidermal growth factor receptor-2 (HER-2) positive rate, sequence of chemotherapy and OFS treatment between the two groups (all P>0.05). The time of menstruation recovery was longer in group A than in group B, the menstruation recovery rate became lower, and the regular rate of menstruation became lower, but there was no significant difference (all P>0.05). After the completion of OFS treatment, the change of AMH and FSH levels in the second year was significantly different from that in the first year ( P=0.003, P=0.004), and the change of AMH and E 2 levels was statistically different between the third year and the second year ( P=0.045, P=0.003). Compared with group B, the serum AMH levels of group A showed decrease after 1, 2 and 3 years, respectively [(0.53±0.15) μg/L vs. (1.01±0.4) μg/L; (0.91±0.25) μg/L vs. (1.38±0.57) μg/L; (1.07±0.23) μg/L vs. (1.55±0.64) μg/L]. Compared with the 1-year serum AMH levels, those of 2-year and 3-year increased 51.5% and 72.1%, respectively. In group A, the serum AMH levels increased 71.7% and 101.9%, respectively, after 2 and 3 years. In group B, the serum AMH levels increased 36.6% and 53.5%, respectively, after 2 and 3 years. The serum E 2, FSH levels were higher or lower in group A than in group B after 1,2, 3 years, showing no significant difference ( P>0.05). Additionally, the differences in serum E 2 levels were statistically significant after 2 years of OFS treatment ( U=520.51,P=0.009). Compared with the 1-year serum E 2 levels, those of 2-year and 3-year decreased 14.8% and increased 59.1%, respectively. In group A, the serum E 2 levels decreased 29.4% and increased 67.6%, respectively, after 2 and 3 years. In group B, the serum E 2 levels decreased 4.4% and increased 52.5%, respectively, after 2 and 3 years. Compared with the 1-year serum FSH levels, those of 2-year and 3-year increased 44.7%, 41.6%, respectively. In group A, the serum FSH levels increased 41.1%, 44.3%, respectively, after 2 and 3 years. In group B, the serum FSH levels increased 48.6%, 47.0%, respectively, after 2 and 3 years. Conclusion:Compared with E 2 and FSH, AMH is more likely to be a used clinically to evaluate the ovarian reserve of breast cancer patients. There is no statistical difference in different OFS courses on the ovarian function of premenstrual breast cancer patients. The ovarian function of patients with the short OFS courses could recover more quickly than those with the long OFS courses. It was necessary to monitor the E 2 levels regularly during OFS treatment.

Objective:To investigate the role of anti-Müllerian hormone (AMH) on the ovarian function evaluation in premenopausal breast cancer patients with hormone receptor positive after different courses of ovarian function suppression (OFS) treatment.Methods:This study was a two-way cohort study. The main endpoints of the study were menstrual recovery and hormone changes after OFS treatment. Totally sixty-seven premenopausal breast cancer patients were enrolled, whose estrogen receptor (ER) and progesterone receptor (PR) had been tested positive from May 2015 to May 2019, and who had undergone operations, chemotherapies and OFS treatments for 24-60 months in Fudan University Shanghai Cancer Center. Twelve cases were eliminated because of missing tracking. The enrolled cases were divided into 2 groups according to OFS treatment, group A was treated with goserelin for 24 months ( n=32), and group B, with goserelin for 25-60 months ( n=23). Following the OFS treatment, the time points of menstrual recovery were observed and the serum levels of AMH, follicle-stimulating hormone (FSH) and estrogen (E 2) levels were measured in the two groups. Results:The follow-up period of the two groups was (42.0±12.3) months. All the patients survived their follow-ups, except one who developed recurrence and metastasis in the 25-60 month course of OFS. No significant differences were observed in age, height, weight, body mass index (BMI), menarche age, pathological type, pathological grade, ER, PR positive rate, human epidermal growth factor receptor-2 (HER-2) positive rate, sequence of chemotherapy and OFS treatment between the two groups (all P>0.05). The time of menstruation recovery was longer in group A than in group B, the menstruation recovery rate became lower, and the regular rate of menstruation became lower, but there was no significant difference (all P>0.05). After the completion of OFS treatment, the change of AMH and FSH levels in the second year was significantly different from that in the first year ( P=0.003, P=0.004), and the change of AMH and E 2 levels was statistically different between the third year and the second year ( P=0.045, P=0.003). Compared with group B, the serum AMH levels of group A showed decrease after 1, 2 and 3 years, respectively [(0.53±0.15) μg/L vs. (1.01±0.4) μg/L; (0.91±0.25) μg/L vs. (1.38±0.57) μg/L; (1.07±0.23) μg/L vs. (1.55±0.64) μg/L]. Compared with the 1-year serum AMH levels, those of 2-year and 3-year increased 51.5% and 72.1%, respectively. In group A, the serum AMH levels increased 71.7% and 101.9%, respectively, after 2 and 3 years. In group B, the serum AMH levels increased 36.6% and 53.5%, respectively, after 2 and 3 years. The serum E 2, FSH levels were higher or lower in group A than in group B after 1,2, 3 years, showing no significant difference ( P>0.05). Additionally, the differences in serum E 2 levels were statistically significant after 2 years of OFS treatment ( U=520.51,P=0.009). Compared with the 1-year serum E 2 levels, those of 2-year and 3-year decreased 14.8% and increased 59.1%, respectively. In group A, the serum E 2 levels decreased 29.4% and increased 67.6%, respectively, after 2 and 3 years. In group B, the serum E 2 levels decreased 4.4% and increased 52.5%, respectively, after 2 and 3 years. Compared with the 1-year serum FSH levels, those of 2-year and 3-year increased 44.7%, 41.6%, respectively. In group A, the serum FSH levels increased 41.1%, 44.3%, respectively, after 2 and 3 years. In group B, the serum FSH levels increased 48.6%, 47.0%, respectively, after 2 and 3 years. Conclusion:Compared with E 2 and FSH, AMH is more likely to be a used clinically to evaluate the ovarian reserve of breast cancer patients. There is no statistical difference in different OFS courses on the ovarian function of premenstrual breast cancer patients. The ovarian function of patients with the short OFS courses could recover more quickly than those with the long OFS courses. It was necessary to monitor the E 2 levels regularly during OFS treatment.

A 55-year-old male patient presented with plaques on the face for more than 20 years,and no immunodeficiency diseases were diagnosed.Skin examination showed large areas of pink plaques on the nose,bilateral cheeks and upper oral lips with slight desquamation,verrucous hyperplasia on the dorsal area of the nose,and a bean-sized verrucous protuberance on the tip of the nose.Histopathological examination of the skin lesions revealed pseudoepitheliomatous hyperplasia in the epidermis and hyphae-like structures in the stratum corneum.Moreover,there was diffuse infiltration of inflammatory cells in the dermis,which mainly included neutrophils,lymphocytes,histiocytes and multinucleated giant cells.Periodic acid-Schiff (PAS)-positive spore-like structures were observed in the multinucleated giant cells.Culture of the lesional tissues on Sabouraud dextrose agar (SDA) medium showed grey-brown villous colonies.Microculture on the potato dextrose agar (PDA) medium yielded dark septate hyphae and pycnidia filled with a large number of spores.Microsphaeropsis arundinis was identified by fungal molecular biological techniques.The patient was diagnosed with cutaneous phaeohyphomycosis caused by Microsphaeropsis arundinis.The patient was treated with CO2 laser for the removal of verrucous protuberance on the tip of the nose,and oral itraconazole capsules at a dose of 200 mg twice a day.After 3-month treatment,the skin lesions subsided and the drug was withdrew.During 6-month follow-up,no relapse occurred.

Objective To investigate the roles of Dectin-1 in phagocytosis of Candida albicans (C.albicans) by macrophage-like cells derived from a human acute monocytic leukemia cell line THP-1.Methods THP-1 macrophage-like cells served as the target cells,and were transfected with small interfering RNA (siRNA) targeting Dectin-1 to down-regulate the expression of Dectin-1 receptor (siRNA-Dectin-1 group).THP-1 macrophage-like cells transfected with nonsense siRNA (siRNA-NC) served as a negative control group.After transfection,the THP-1 macrophage-like cells in the above 2 groups were cocultured with heat-killed C.albicans separately.And then,fluorescence microscopy was performed to count THP-1 macrophage-like cells phagocytosing C.albicans,and flow cytometry was used to determine the mean fluorescence intensity (MFI) of dihydrorhodamine (DHR)-123 fluorescent cells.Statistical analysis was done by one-way analysis of variance (ANOVA) and t test with the SPSS19.0 software.Results After transfection with siRNA-Dectin-1,the mRNA and protein expression of Dectin-1 significantly decreased in THP-1 macrophage-like cells (t =26.163,P ＜ 0.001).After 1-,2-,4-hour co-culture of THP-1 macrophagelike cells with C.albicans,fluorescence microscopy showed that the phagocytosis rates of C.albicans by THP -1 macrophage-like cells were significantly lower in the siRNA-Dectin-1 group than in the negative control group (17.5％ vs.22.1％,18.6％ vs.24.3％,39.2％ vs.59.1％,respectively,all P ＜ 0.05),so were the percentage of THP-1 macrophage-like cells phagocytosing more than 3 C.albicans cells (2.2％ vs.4.7％,2.5％ vs.5.4％,5.1％ vs.8.3％,respectively,all P ＜ 0.05).After 30-minute,1-,2-and 4-hour co-culture of THP-1 macrophage-like cells with DHR-123-labelled C.albicans,flow cytometry showed that the MFI of C.albicans-phagocytosing cells was significantly lower in the siRNA-Dectin-1 group than in the negative control group (36.8 vs.45.7,54.3 vs.62.4,72.1 vs.84.9,93.6 vs.116.7,respectively,all P ＜ 0.05).Conclusion Dectin-1 receptor plays an important role in the phagocytosis of C.albicans by macrophages.

Objective:To investigate the biological characteristics of monoclonal antibodies against avian influenza virus (AIV).Methods:Monoclonal antibodies (mAbs) against AIV H5N1 were prepared and it′s characteristics were identified including subtype,titer,hemagglutination inhibition activity and cross-reactivity with other influenza viruses.Besides,Western blot and immuno-histochemical staining methods were conducted to test the combination of antibodies and antigen ( H5N1 ) and human normal tissues.Results:Immunohistochemical analysis showed that 2 mAbs (H5-32 and H5-35) cross-reacted with human tissues kidney and pancreas respectively.Conclusion:These data indicated that there have some association between the AIV H 5N1 with human tissues, which may provide reference for the study on avian influenza virus infection and pathogenicity .

Objective To evaluate effects of the yeast form of Sporothrix schenckii on activation of p38 mitogen-activated protein kinase (p38MAPK) and expression of interleukin-6 (IL-6) in macrophagelike THP-1 cells,which were differentiated from the human acute monocytic leukemia cell line THP-1.Methods THP-1 macrophage-like cells were divided into 3 groups to be treated with the yeast form of Sporothrix schenckii at a concentration of 2 × 106 colony-forming units (CFU)/ml (yeast form group),100 mg/L curdlan (curdlan group) and RPMI 1640 medium (blank control group) respectively.Real-time fluorescence-based quantitative PCR was performed to measure the mRNA expression of IL-6 in THP-1 macrophage-like cells in the above 3 groups after 3-and 6-hour treatment separately,and enzyme-linked immunosorbent assay (ELISA) to detect the level of IL-6 in the culture supernatant of THP-1 macrophagelike cells after 24-hour treatment.Western blot analysis was conducted to determine the protein expression of p38MAPK and phosphorylated p38MAPK (p-p38MAPK) in the above 3 groups after 30-and 60-minute treatment separately.Other THP-1 macrophage-like cells were pretreated with 100 nmol/L dexamcthasonc (a p38MAPK inhibitor) for 30 minutes,and then were divided into 3 groups to be treated with the yeast form of Sporothrix schenckii,curdlan and RPMI 1640 medium respectively,and changes in the level of pp38MAPK and mRNA expression of IL-6 were also detected.Statistical analysis was carried out with SPSS19.0 software by using one-way or multi-way analysis of variance and least significant difference (LSD) test.Results Significant differences in the mRNA expression of IL-6 in THP-1 macrophage-like cells were observed among the yeast form group,curdlan group and blank control group (F =5 552.22,P ＜0.001) after 3-hour treatment (56.81 ± 7.36,26.69 ± 1.22 and 0.97 ± 0.05,respectively) and 6-hour treatment (378.03 ± 16.67,276.24 ± 39.13 and 1.02 ± 0.04,respectively).Additionally,the yeast form group showed significantly higher mRNA expression of IL-6 after 6-hour treatment than that after 3-hour treatment (q =16.74,P ＜ 0.001).After 24-hour treatment,the level of IL-6 in the culture supernatant of THP-1 macrophage-like cells also significantly differed among the yeast form group,curdlan group and blank control group (59.96 ± 18.16 pg/L,91.01 ± 17.27 pg/L,5.50 ± 2.30 pg/L,respectively;F =26.62,P ＜ 0.01),and was significantly higher in the yeast form group than in the blank control group (P ＜ 0.01).After 30-and 60-minute treatment,the protein expression of p-p38MAPK was significantly higher in the yeast form group than in the blank control group (both P ＜ 0.01).Moreover,the mRNA expression of IL-6 (4.46 ± 1.03 vs.493.52 ± 113.87,P ＜ 0.001) and protein expression of p-p38MAPK (2.29 ± 0.37 vs.4.55 ±0.46,q =10.81,P ＜ 0.01) were both significantly lower in the yeast form group with dexamethasone pretreatment than in that without dexamethasone pretreatment.Conclusion In vitro treatment with the yeast form of Sporothrix schenckii can enhance the expression of IL-6 in human THP-1 macrophage-like cells by activating the p38MAPK signaling pathway.

Objective To evaluate the in vitro synergistic effect of tetrandrine on ketoconazole against Candida parapsilosis complex.Methods According to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines,the microdilution checkerboard method was used to evaluate in vitro antifungal activities of ketoconazole alone and in combination with tetrandrine against 21 clinical isolates of Candida parapsilosis complex based on the fractional inhibitory concentration index (FICI).Antifungal effects of the above drugs at different time points were evaluated by the XTT assay,and then time-killing curves were drawn and assessed to investigate the in vitro dynamic antifungal activity.Results The minimum inhibitory concentrations (MICs) of tetrandrine and ketoconazole alone against 21 clinical isolates of Candida parapsilosis complex were 32-64 mg/L and 0.031 25-2 mg/L,respectively.When ketoconazole was combined with tetrandrine,MICs of tetrandrine and ketoconazole were reduced to 2-8 mg/L and 0.008-0.25 mg/L respectively,and the FICI ranged from 0.09 to 0.5.The time-killing curves revealed that the fungal growth was delayed obviously in the combination group compared with the ketoconazole alone group and tetrandrine alone group.Conclusion Tetrandrine has obvious synergistic effects on ketoconazole against Candida parapsilosis complex in vitro.

Objective Trichophyton rubrum strains can cause superficial infection and also deep tissue infection, but relevant animal model has not been reported yet.The aim of this study was to construct an animal model of granuloma infected by T.rubrum. Methods Three T.rubrum strains isolated from clinical granuloma tissues, 2 T.rubrum strains isolated from tinea infection and a standard strain of ATCCMYA4438 were selected.Corticosteroids were given to the Balb/C mice before and after the injection of the T. rubrum and mucin suspension and the mice model of granuloma was established.Direct microcopy, culture and histopathologic method were adopt to verify the infection effects. Results The mice inoculated with the T.rubrum granuloma strains with mucin suspension were examined after 21 days in the condition of applying appropriate dose of glucocorticoids.Direct microscopic examination showed the slender mycelium, fungal culture showed the growth of colony and histopathology showed excessive keratinization of foot tissue, formation of granuloma in the dermis with inflammatory cell infiltration of neutro-philic granulocyte and lymphocytes.However, the mice inoculated with the T.Rubrum tinea strains with mucin suspension showed the negative result. Conclusion The rubrum granuloma mice model can be es-tablished using the clinical isolates of T.rubrum granuloma strains with the mucin and glucocorticoids interventions.

A 76?year?old female patient complained of right chest pain for three months. CT scan showed a clump?like high?density shadow measuring 4.8 cm × 3.0 cm in size in the dorsal portion of the right lower lobe of the lung. Aspiration biopsy was performed, and biopsy samples were subjected to fungal culture and histopathological examination. Histopathological examination showed chronic granulomatous inflammation with hyaline septate hyphae. After 4?day culture, white villous dense colonies were formed on the Sabouraud′s agar medium. The center of the colonies was slightly elevated with wrinkles or radiating striae on the surface, and the bottom of the colonies was faint yellow in color. Microculture yielded abundant septate branched hyphae, and very few colorless hyaline quasi?circular spores. DNA sequencing of rDNA internal transcribed spacer (ITS) regions and β?tubulin genes was performed to identify the isolate, and antifungal susceptibility testing was carried out in vitro. The MEGA7.0 software was used to build phylogenetic trees of Aspergillus fumigatus complex and its closely related species. The isolate was identified as Aspergillus fumigatus by molecular biologic sequencing. The patient was diagnosed with pulmonary aspergilloma. After administration of itraconazole oral solution and vorionazole tablets, the condition got better obviously.