Objective·To determine hyaluronan(HA)expression in the endocrine-resistant microenvironment of estrogen receptor-positive(ER+)breast cancer and elucidate its impact on the acquired resistance.Methods·Chemiluminescent immunoassay was used to quantify HA levels in the culture supernatants of fulvestrant-resistant breast cancer cells.An immunofluorescence(IF)assay was performed to visualize the colocalization of CD44 and HA in MCF7/FulR cells.Using an established adaptive endocrine-resistant breast cancer mouse model,HA expression in resistant breast cancer tissues was assessed by immunohistochemistry(IHC)assay.Single-cell RNA sequencing(scRNA-seq)and RNA sequencing(RNA-seq)were conducted to examine transcriptomic profiles and alterations in HA-related genes in resistant breast cancer cells.Flow cytometry(FCM)was utilized to measure the proportion of CD44+CD24-cells in MCF7/FulR.The correlation between HA synthesis genes and cell stemness was investigated in clinical ER+breast cancers from GEO data sets.Hyaluronidase(HAase)treatment was applied to remove the"HA coat",and RT-qPCR and Western blotting analysis were carried out to monitor changes in stemness-related molecules.CCK-8 assays,flow cytometry(FCM),and Hoechst 33258 staining were performed to determine changes in apoptosis and fulvestrant efficiency after HAase treatment.Results·IF results revealed that compared with MCF7 cells,the"HA coat"on the surface of MCF7/FulR cells was significantly thickened.IHC demonstrated markedly increased HA retention in fulvestrant-resistant mouse breast cancer tissues.ScRNA-seq and RNA-seq analyses indicated elevated expression of stemness-related genes and HA synthesis-associated genes in fulvestrant-resistant breast cancer cells.Correlation analysis revealed a positive association between HA synthesis and cancer stemness in ER+breast cancer.IF and RT-qPCR results demonstrated that removing the HA coating from the surface of MCF7/FulR cells led to a significant reduction in the expression of stemness-related molecules;concurrently,CCK-8 assays,FCM analysis,and Hoechst 33258 staining revealed that"HA coat"clearance reduced MCF7/FulR'tolerance to fulvestrant and increased apoptosis.Conclusion·Endocrine-resistant breast cancer cells develop an enriched"HA coat",which promotes stemness in fulvestrant-resistant tumors.Disruption of this HA coat through HAase treatment effectively reduces cell stemness,induces apoptosis,and re-sensitizes breast cancer cells to fulvestrant.

Objective·To determine hyaluronan(HA)expression in the endocrine-resistant microenvironment of estrogen receptor-positive(ER+)breast cancer and elucidate its impact on the acquired resistance.Methods·Chemiluminescent immunoassay was used to quantify HA levels in the culture supernatants of fulvestrant-resistant breast cancer cells.An immunofluorescence(IF)assay was performed to visualize the colocalization of CD44 and HA in MCF7/FulR cells.Using an established adaptive endocrine-resistant breast cancer mouse model,HA expression in resistant breast cancer tissues was assessed by immunohistochemistry(IHC)assay.Single-cell RNA sequencing(scRNA-seq)and RNA sequencing(RNA-seq)were conducted to examine transcriptomic profiles and alterations in HA-related genes in resistant breast cancer cells.Flow cytometry(FCM)was utilized to measure the proportion of CD44+CD24-cells in MCF7/FulR.The correlation between HA synthesis genes and cell stemness was investigated in clinical ER+breast cancers from GEO data sets.Hyaluronidase(HAase)treatment was applied to remove the"HA coat",and RT-qPCR and Western blotting analysis were carried out to monitor changes in stemness-related molecules.CCK-8 assays,flow cytometry(FCM),and Hoechst 33258 staining were performed to determine changes in apoptosis and fulvestrant efficiency after HAase treatment.Results·IF results revealed that compared with MCF7 cells,the"HA coat"on the surface of MCF7/FulR cells was significantly thickened.IHC demonstrated markedly increased HA retention in fulvestrant-resistant mouse breast cancer tissues.ScRNA-seq and RNA-seq analyses indicated elevated expression of stemness-related genes and HA synthesis-associated genes in fulvestrant-resistant breast cancer cells.Correlation analysis revealed a positive association between HA synthesis and cancer stemness in ER+breast cancer.IF and RT-qPCR results demonstrated that removing the HA coating from the surface of MCF7/FulR cells led to a significant reduction in the expression of stemness-related molecules;concurrently,CCK-8 assays,FCM analysis,and Hoechst 33258 staining revealed that"HA coat"clearance reduced MCF7/FulR'tolerance to fulvestrant and increased apoptosis.Conclusion·Endocrine-resistant breast cancer cells develop an enriched"HA coat",which promotes stemness in fulvestrant-resistant tumors.Disruption of this HA coat through HAase treatment effectively reduces cell stemness,induces apoptosis,and re-sensitizes breast cancer cells to fulvestrant.

Objective To explore the correlation between childhood trauma and plasma adiponectin levels in patients with depression.Methods A total of 121 patients with depression and 39 healthy controls(control group)were enrolled.Childhood trauma questionnaire(CTQ-SF)was used to assess the experience of childhood abuse and neglect,and the patients with depression were divided into trauma group(n=53)and non-trauma group(n=68)according to the CTQ-SF score.The 17-item Hamilton depression scale-17(HAMD17)and the Hamilton anxiety scale(HAMA)were used to evaluate the severity of depression and anxiety symptoms,respectively.Plasma adiponectin levels of subjects were measured by enzyme-linked immunosorbent assay.Results The plasma adiponectin level of trauma group[3.82(2.44,4.92)μg/mL]was significantly lower than that of non-trauma group[4.64(2.98,6.43)μg/mL,P=0.01]and the control group[6.29(4.54,7.51)μg/mL,P＜0.01].The plasma adiponectin level of non-trauma group was lower than that of the control group(P＜0.01).Correlation analysis showed that plasma adiponectin level in patients with depression was negatively correlated with childhood trauma(r=-0.34,P＜0.01).Multivariate linear regression analysis showed that plasma adiponectin level was negatively correlated with childhood trauma scores in patients with depression(β=-0.05,P＜0.01).Conclusions Patients with depression who have experienced childhood trauma have lower plasma levels of adiponectin,and childhood trauma may be associated with decreased plasma adiponectin levels in patients with depression.

Objective:To observe the effect of computerized cognitive remediation therapy(CCRT) in the patients with mild cognitive impairment (MCI).Methods:A randomized, single-blinded clinical study was carried out from the April to June in 2019. 46 patients who met MCI criteria were randomly allocated into a CCRT group ( n=24) and a control group ( n=22). In CCRT group, the CCRT was conducted five times a week (30 minutes each time) for a total of 8 weeks (40 times), while a natural observation was performed in the control group. All the subjects were assessed by the Mini-Mental State Examination(MMSE) and the Montreal Cognitive Assessment(MoCA) before and after the treatment. The Wilcoxon test in the paired rank-sum test of two related samples was used to evaluate the effect of CCRT on MCI before and after the intervention, and the Mann-Whitney U test in the rank-sum test of two independent samples was used to compare the differences in MMSE and MoCA scores between the two groups. Results:Before treatment, there were no statistically significant differences in MMSE, MoCA total scores and each factor between the CCRT group and the control group ( P>0.05). A total of 21 patients in CCRT group completed CCRT treatment. After 8 weeks of treatment, the difference between two groups in the total score of MMSE ( Z=-2.83), attention and calculation( Z=-2.58), time orientation( Z=-2.00) and visual spatial function ( Z=-2.45) scores were higher than those before the treatment ( P<0.05); the difference between two groups in MoCA total score ( Z=-3.40), visual space and executive function( Z=-3.41), attention ( Z=-3.09) were higher than those before the treatment ( P<0.05). Conclusion:CCRT may improve the cognitive function of MCI patients, especially the attention and visuospatial functions.

Objective:To observe the effect of computerized cognitive remediation therapy(CCRT) in the patients with mild cognitive impairment (MCI).Methods:A randomized, single-blinded clinical study was carried out from the April to June in 2019. 46 patients who met MCI criteria were randomly allocated into a CCRT group ( n=24) and a control group ( n=22). In CCRT group, the CCRT was conducted five times a week (30 minutes each time) for a total of 8 weeks (40 times), while a natural observation was performed in the control group. All the subjects were assessed by the Mini-Mental State Examination(MMSE) and the Montreal Cognitive Assessment(MoCA) before and after the treatment. The Wilcoxon test in the paired rank-sum test of two related samples was used to evaluate the effect of CCRT on MCI before and after the intervention, and the Mann-Whitney U test in the rank-sum test of two independent samples was used to compare the differences in MMSE and MoCA scores between the two groups. Results:Before treatment, there were no statistically significant differences in MMSE, MoCA total scores and each factor between the CCRT group and the control group ( P>0.05). A total of 21 patients in CCRT group completed CCRT treatment. After 8 weeks of treatment, the difference between two groups in the total score of MMSE ( Z=-2.83), attention and calculation( Z=-2.58), time orientation( Z=-2.00) and visual spatial function ( Z=-2.45) scores were higher than those before the treatment ( P<0.05); the difference between two groups in MoCA total score ( Z=-3.40), visual space and executive function( Z=-3.41), attention ( Z=-3.09) were higher than those before the treatment ( P<0.05). Conclusion:CCRT may improve the cognitive function of MCI patients, especially the attention and visuospatial functions.

Objective:To explore the effects and related mechanisms of curcumin and resveratrol on macrophages of colon tissues in ulcerative colitis (UC) mice.Methods:Sixty BALB/c mice were randomly and equally divided into CON group, DSS group, DSS+Cur group, DSS+Res group, Eve+Cur group and Eve+Res group. There were 10 mice in each group. CON group was the normal control group and received no treatment. UC model of mice was established by giving 3% dextran sodium sulfate (DSS) to drink freely for 7 days in other 5 groups. UC model of mice was simply established in DSS group. The gavage administrations of 0.4 ml curcumin and 0.4 ml resveratrol were performed every day in the mice of DSS+Cur group and DSS+Res group respectively after establishing the UC model. The gavage administration of 0.4 ml mTOR inhibitor Everolimus was performed in Eve+Cur group after 0.4 ml curcumin gavage administration and also was performed in Eve+Res group after 0.4 ml resveratrol gavage administration every day. The mice were sacrificed after 7 days of administration. The macrophages of colon tissues were isolated and cultured in each group. The general conditions of the mice were observed and the DAI score was evaluated. In order to examine the effects of curcumin and resveratrol on macrophages, CCK-8 method was used to detect the proliferation and flow cytometry was used to detect the apoptosis of macrophages. Fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expressions of the autophagy-related factors LC3, Beclin-1 and Atg12. ELISA was used to detect the level of the inflammatory factors IL-6 and TNF-α in culture supernatants of macrophages. In order to explore the associated mechanism, ELISA was used to detect the influence of mTOR inhibitor Everolimus on IL-6 and TNF-α, fluorescence quantitative PCR and Western blot were used to detect the expressions of SIRT1/mTOR pathway-related factors mTOR and SIRT1 mRNA and protein.Results:No death was observed in all of the mice. Compared with CON group, the proliferative activity of macrophages was significantly higher, and the apoptosis rate decreased significantly, the mRNA and protein expressions of Atg12, Beclin-1 and LC3 increased significantly in DSS Group, DSS+Cur group and DSS+Res group (all P<0.05) . Compared with DSS group, the proliferative activity was significantly lower, the apoptosis rate increased obviously, the mRNA and protein expressions of Atg12, Beclin-1 and LC3 decreased significantly in DSS+Cur group and DSS+Res group (all P<0.05) . Compared with CON group, the DAI scores increased, the mRNA and protein expressions of mTOR and SIRT1 decreased significantly, the levels of IL-6 and TNF-α increased significantly in DSS group, DSS+Cur group, DSS+Res group, Eve+Cur group and Eve+Res group (all P<0.05) . Compared with DSS group, the DAI scores decreased, the mRNA and protein expressions of mTOR and SIRT1 increased significantly, the levels of IL-6 and TNF-α decreased significantly in DSS+Cur group and DSS+Res group (all P<0.05) . Compared with DSS+Cur group and DSS+Res Group, the DAI scores increased significantly, the mRNA and protein expressions of mTOR and SIRT1 decreased significantly, the levels of IL-6 and TNF-α increased significantly in Eve+Cur group and Eve+Res group (all P<0.05) . Conclusion:Curcumin and resveratrol can inhibit the macrophages proliferation of colon tissues in UC mice, promote the apoptosis and inhibit the development of inflammation and autophagy, which may be regulated by SIRT1/mTOR pathway.

Objective:To explore the effects and related mechanisms of curcumin and resveratrol on macrophages of colon tissues in ulcerative colitis (UC) mice.Methods:Sixty BALB/c mice were randomly and equally divided into CON group, DSS group, DSS+Cur group, DSS+Res group, Eve+Cur group and Eve+Res group. There were 10 mice in each group. CON group was the normal control group and received no treatment. UC model of mice was established by giving 3% dextran sodium sulfate (DSS) to drink freely for 7 days in other 5 groups. UC model of mice was simply established in DSS group. The gavage administrations of 0.4 ml curcumin and 0.4 ml resveratrol were performed every day in the mice of DSS+Cur group and DSS+Res group respectively after establishing the UC model. The gavage administration of 0.4 ml mTOR inhibitor Everolimus was performed in Eve+Cur group after 0.4 ml curcumin gavage administration and also was performed in Eve+Res group after 0.4 ml resveratrol gavage administration every day. The mice were sacrificed after 7 days of administration. The macrophages of colon tissues were isolated and cultured in each group. The general conditions of the mice were observed and the DAI score was evaluated. In order to examine the effects of curcumin and resveratrol on macrophages, CCK-8 method was used to detect the proliferation and flow cytometry was used to detect the apoptosis of macrophages. Fluorescence quantitative PCR and Western blot were used to detect the mRNA and protein expressions of the autophagy-related factors LC3, Beclin-1 and Atg12. ELISA was used to detect the level of the inflammatory factors IL-6 and TNF-α in culture supernatants of macrophages. In order to explore the associated mechanism, ELISA was used to detect the influence of mTOR inhibitor Everolimus on IL-6 and TNF-α, fluorescence quantitative PCR and Western blot were used to detect the expressions of SIRT1/mTOR pathway-related factors mTOR and SIRT1 mRNA and protein.Results:No death was observed in all of the mice. Compared with CON group, the proliferative activity of macrophages was significantly higher, and the apoptosis rate decreased significantly, the mRNA and protein expressions of Atg12, Beclin-1 and LC3 increased significantly in DSS Group, DSS+Cur group and DSS+Res group (all P<0.05) . Compared with DSS group, the proliferative activity was significantly lower, the apoptosis rate increased obviously, the mRNA and protein expressions of Atg12, Beclin-1 and LC3 decreased significantly in DSS+Cur group and DSS+Res group (all P<0.05) . Compared with CON group, the DAI scores increased, the mRNA and protein expressions of mTOR and SIRT1 decreased significantly, the levels of IL-6 and TNF-α increased significantly in DSS group, DSS+Cur group, DSS+Res group, Eve+Cur group and Eve+Res group (all P<0.05) . Compared with DSS group, the DAI scores decreased, the mRNA and protein expressions of mTOR and SIRT1 increased significantly, the levels of IL-6 and TNF-α decreased significantly in DSS+Cur group and DSS+Res group (all P<0.05) . Compared with DSS+Cur group and DSS+Res Group, the DAI scores increased significantly, the mRNA and protein expressions of mTOR and SIRT1 decreased significantly, the levels of IL-6 and TNF-α increased significantly in Eve+Cur group and Eve+Res group (all P<0.05) . Conclusion:Curcumin and resveratrol can inhibit the macrophages proliferation of colon tissues in UC mice, promote the apoptosis and inhibit the development of inflammation and autophagy, which may be regulated by SIRT1/mTOR pathway.

Objective To observe the changes of adiponectin (APN),chemerin and tumor necrosis factor-α (TNF-α) in subclinical hypothyroidism (SCH) rats,and to clarify the effect of L-thyroxine (L-T4) replacement therapy.Methods Sixty-five male Wistar rats were randomly divided into five groups via the random number table method:control group (n =10),SCH group A (n =15),SCH group B (n =15),SCH group C (n =15) and L-T4 treatment group (SCH + L-T4,n =10).Rats in groups SCH A,B and C were fed with 5,15 and 20 mg·kg-1·d-1 methimazole (MMI) once daily by gavage.The rats in SCH + L-T4 group were given 20 mg·kg-1·d-1 MMI once daily through gavage,after 8 weeks,6 μg·kg-1· d-1 of L-T4 was intragastrically added (50 μg/tablet) and the model was completed at the 16th week.The levels of serum APN,chemerin and TNF-α were measured via the enzyme linked immunosorbent assay (ELISA) method.The mRNA and protein levels of APN,chemerin and TNF-α in visceral adipose tissue of 5 groups were determined by real-time PCR (RT-PCR) and Western blotting,respectively.Results Compared with the control group [(202.20 + 17.27) ng/L,(143.70 ± 18.46) ng/L,(114.69 ± 4.18) μg/L],the serum chemerin levels in the SCH A,B,C groups were significantly higher [(314.33 ± 16.80),(355.00 ± 17.10),(365.00 ± 11.63) ng/L,P ＜0.05] and TNF-α levels also increased significantly [(222.60 ± 14.13),(279.20 ± 12.79),(288.30 ± 15.89) ng/L,P ＜0.05],and APN levels were significantly decreased [(77.21 ± 3.08),(68.58 ± 2.92),(59.45 ± 2.41) μg/L,P ＜0.05];but compared with SCH group C,the levels of chemerin and TNF-α in the SCH + L-T4 group were decreased [(260.07 ± 10.80),(178.40 ± 10.29) ng/L] and the level of APN [(102.35 ± 3.17) μg/L] was increased (P＜ 0.05).The mRNA and protein levels of APN in SCH A,B,C groups were significantly lower than those in the control group (P ＜0.05).The APN mRNA and protein levels in the SCH + L-T4 group were significantly higher than those in the SCH group C (P ＜ 0.05).The mRNA and protein levels of chemerin and TNF-α in the SCH A,B,C groups were higher than those in the control group (P ＜ 0.05).However,the mRNA and protein levels of chemerin and TNF-α in the SCH + L-T4 group were significantly lower than those in the SCH group C (P ＜ 0.05).Conclusion The expression levels of serum chemerin and TNF-α in SCH rats have increased,and APN levels decreased,but L-T4 can ameliorate these changes.

Objective To compare the results of TEOAE and DPOAE in the same population of normal newborns, to provide information on choosing appropriate screening tools.Methods A two-steps protocol was taken with the first screening during the first 48 to 72 hours of birth and rescreened from one to two months old if the newborns failed the first screening.For each step of screening, TEOAE and DPOAE were performed simultaneously using AccuScreen hearing screening instrument (Madsen-GN Otometrics, Taastrup, Denmark).A total of 1 062 normal newborns (F/M=508/554) delivered in Peking Union Medical College Hospital were enrolled in this research for the first screening.Infants who failed either TEOAE or DPOAE screening in the first screening were referred to a second screening.Among them, 135 performed both DPOAE and TEOAE in the second step.The newborns who failed the second screening would receive ABR when they were 3 months old.Results In the first screening,the failure rate for TEOAE was 11.0% (117/1 062) and 13.7% (145/1 062) for DPOAE.In the second screening step, the failure rates were 17.8% (24/135) and 20.7% (28/135) for TEOAE and DPOAE, respectively.Chi-square and Fisher's test showed that the failure rates of DPOAE were significant higher than TEOAE for both steps (P<0.001).The agreements between TEOAE and DPOAE were 96.0% and 95.6% for the first and second steps respectively, and the kappa values were 0.817 and 0.857.As to the average time taken to accomplish the screening for one ear, TEOAE was 24±25 s and DPOAE was 40±34 s during the first screening;in the rescreening, TEOAE was 52±41 s and DPOAE was 73±62 s.Paired-t tests showed that the differences between DPOAE and TEOAE testing time were statistically significant (P=0.000) in both screening steps.Finally, 7 newborns (10 ears) were diagnosed conductive hearing loss(except 1 ear was sensorineural hearing loss).Conclusion As a screening tool, TEOAE got lower refer rates and took less time than DPOAE implicating TEOAE a better screening tool for normal neonates.

Objective To investigate the application of radial endobronchial ultrasound (R-EBUS)-guided bronchoscopy in the diagnosis of peripheral pulmonary lesions (PPLs),assess its safety and diagnostic value,and explore the influencing factors.Methods Clinical data of 140 patients who underwent R-EBUS transbronchial biopsy (TBB) and brush biopsy in our department of respiratory endoscopy from January 2015 to March 2017 were collected and retrospectively analyzed in the study.Eighty-three cases were diagnosed as PPLs.The detection rate,ability to locate the peripheral lesions,and influencing factors of EBUS were analyzed.The incidence rate of complications was observed to assess its safety.Results Of the 83 PPLs patients,they were 55 males and 28 females,and at a mean age of 59.81 ± 11.85 years.The total success rate of EBUS-guided bronchoscopic diagnosis was 59.04％ (49/83),and the diagnostic rate was 50.94％ (27/53) for malignant lesions,and 73.33％ (22/30) for benign diseases.The rate of EBUS-TBB (56.92％,37/65) was significantly higher than that of brush biopsy (30.12％,25/83,Chi square =10.76,P =0.001).EBUS-guided diagnostic methods had an accuracy of 60.00％ (45/75),while the EBUS-guided bronchoscopy had a rate of 50.0％ (4/8),but no significant difference was seen between the 2 methods (Chi square =0.03,P =0.87).The sizes of the lesions ranged from 10.0 to 52.4 mm,and the diagnostic yield for PPLs ≤20 mm in diameter (36.84％,7/19) was lower than that for those ＞20 mm (65.62％,42/64,Chi square =5.02,P =0.003).There was no significant difference (Chi square =10.05,P =0.07)in the diagnostic yield for different sites,with that of right upper lobe of 46.15％ (12/26),right middle lobe 100.00％ (8/8),right lower lobe 53.85％ (7/13),left upper lobe 37.50％ (3/8),left lingula lobe 63.64％ (7/11),left lower lobe 70.59％ (12/17).But the detection rate was obviously lower in the upper lobe (22/45,48.89％) than the middle/lower lobe (27/38,71.05％,Chi square =5.02,P =0.003).Of all the 83 patients,74 ones (89.16％) were successfully identified using radial probe EBUS,but the lesions were not found in 9 patients.When the radial probe position was within the target lesion,the diagnostic yield was 81.58％ (31/38),notably higher than that when the probe was positioned adjacent to the lesion (50.00％,18/36,Chi square =8.24,P =0.004).Mild bleeding was observed when performing biopsy under bronchoscope,and no pneumothorax,or other serious complications were observed.Conclusion Radial EBUS is a safe and feasible nethod to accurately identify PPLs and improve its diagnostic rate.EBUS-guided bronchoscopy has higher positive detection rate for PPLs,and is cost saving.The relationship of the probe and the site of lesion is the main factor influencing the diagnostic rate.