Single-Cell Analyses Reveal Mechanisms of Cancer Stem Cell Maintenance and Epithelial-Mesenchymal Transition in Recurrent Bladder Cancer.

PURPOSE: Bladder cancer treatment remains a major clinical challenge due to therapy resistance and a high recurrence rate. Profiling intratumor heterogeneity can reveal the molecular mechanism of bladder cancer recurrence. EXPERIMENTAL DESIGN: Here, we performed single-cell RNA sequencing and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on tumors from 13 patients with low recurrence risk, high recurrence risk, and recurrent bladder cancer. RESULTS: Our study generated a comprehensive cancer-cell atlas consisting of 54,971 single cells and identified distinct cell subpopulations. We found that the cancer stem-cell subpopulation is enriched during bladder cancer recurrence with elevated expression of EZH2. We further defined a subpopulation-specific molecular mechanism whereby EZH2 maintains H3K27me3-mediated repression of the NCAM1 gene, thereby inactivating the cell invasive and stemness transcriptional program. Furthermore, taking advantage of this large single-cell dataset, we elucidated the spectrum of epithelial-mesenchymal transition (EMT) in clinical samples and revealed distinct EMT features associated with bladder cancer subtypes. We identified that TCF7 promotes EMT in corroboration with single-cell ATAC with high-throughput sequencing (scATAC-seq) analysis. Additionally, we constructed regulatory networks specific to recurrent bladder cancer. CONCLUSIONS: Our study and analytic approaches herein provide a rich resource for the further study of cancer stem cells and EMT in the bladder cancer research field.

Diffusion Tensor Magnetic Resonance Imaging for Differentiating Multiple System Atrophy Cerebellar Type and Spinocerebellar Ataxia Type 3.

Multiple system atrophy cerebellar type (MSA-C) and spinocerebellar ataxia type 3 (SCA3) demonstrate similar manifestations, including ataxia, pyramidal and extrapyramidal signs, as well as atrophy and signal intensity changes in the cerebellum and brainstem. MSA-C and SCA3 cannot be clinically differentiated through T1-weighted magnetic resonance imaging (MRI) alone; therefore, clinical consensus criteria and genetic testing are also required. Here, we used diffusion tensor imaging (DTI) to measure water molecular diffusion of white matter and investigate the difference between MSA-C and SCA3. Four measurements were calculated from DTI images, including fractional anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD). Fifteen patients with MSA-C, 15 patients with SCA3, and 30 healthy individuals participated in this study. Both patient groups demonstrated a significantly decreased FA but a significantly increased AD, RD, and MD in the cerebello-ponto-cerebral tracts. Moreover, patients with SCA3 demonstrated a significant decrease in FA but more significant increases in AD, RD, and MD in the cerebello-cerebral tracts than patients with MSAC. Our results may suggest that FA and MD can be effectively used for differentiating SCA3 and MSA-C, both of which are cerebellar ataxias and have many common atrophied regions in the cerebral and cerebellar cortex.

Correction to: Prognostic Role of the Immunoscore for Patients with Urothelial Carcinoma of the Bladder Who Underwent Radical Cystectomy.

In the original article, the word IMMUNOSCORE® was not displayed to reflect its trademark status. At every mention, IMMUNOSCORE® should be in all caps and with a registered trademark symbol.

Prognostic Role of the Immunoscore for Patients with Urothelial Carcinoma of the Bladder Who Underwent Radical Cystectomy.

BACKGROUND: Increasing evidence suggests that cancer progression is strongly influenced by the host immune response, which is represented by immune cell infiltrates. The T-lymphocyte-based Immunoscore is reported to be a reliable prognostic factor in colon cancer, but its significance in urothelial carcinoma of the bladder (UCB) is at an early stage of exploration. This study aimed to determine whether the tumor immune infiltrate, as evaluated by the Immunoscore, could act as a useful prognostic marker for UCB patients who have undergone radical cystectomy (RC). METHODS: In this study, immunohistochemistry was used to examine the Immunoscore of 221 UCB patients who underwent RC. The Immunoscore of the patients was determined by the densities of CD3+ and CD8+ T cells at the tumor center and the invasive margin. RESULTS: A highly significant association between a low Immunoscore and a shortened patient survival (P < 0.001, log-rank test) was demonstrated. In different subsets of UCB patients, a low Immunoscore also was a prognostic indicator of pT ≤ 2, pN(-)-status tumors, negative vascular invasion, or both (P < 0.05). Importantly, the Immunoscore together with the patient's pT status provided significant independent prognostic parameters in the multivariate analysis (P < 0.05). Furthermore, a significant correlation (P = 0.003) of a low Immunoscore with an increased UCB labeling index of Ki-67 (a cell proliferation marker) was observed in this UCB cohort. CONCLUSIONS: The findings suggest that the Immunoscore, as examined by immunohistochemistry, might serve as a novel prognostic marker for UCB patients who have undergone RC.

Extracellular heat shock protein 90α mediates HDM-induced bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling.

BACKGROUND: The disruption and hyperpermeability of bronchial epithelial barrier are closely related to the pathogenesis of asthma. House dust mite (HDM), one of the most important allergens, could increase the airway epithelial permeability. Heat shock protein (Hsp) 90α is also implicated in the lung endothelial barrier dysfunction by disrupting RhoA signaling. However, the effect of extracellular Hsp90α (eHsp90α) on the bronchial epithelial barrier disruption induced by HDM has never been reported. METHODS: To investigate the involvement of eHsp90α in the bronchial epithelial barrier disruption induced by HDM, normal human bronchial epithelial cell line 16HBE14o- (16HBE) cells were treated by HDM, human recombinant (hr) Hsp90α and hrHsp90ß respectively and pretreated by1G6-D7, a specific anti-secreted Hsp90α monoclonal antibody (mAb). Hsp90α-silencing cells were also constructed. To further evaluate the role of RhoA signaling in this process, cells were pretreated by inhibitors of Rho kinase, GSK429286A and Y27632 2HCl. Transepithelial electrical resistance (TEER) and FITC-dextran flux (FITC-DX) were examined as the epithelial barrier function. Expression and localization of adherens junctional proteins E-cadherin and ß-catenin were evaluated by western blotting and immunofluorescence respectively. The level of eHsp90α was investigated by concentration and purification of condition media. RhoA activity was determined by using a Rho G-LISA® RhoA activation assay kitTM biochem kit, and the phosphorylation of myosin light chain (MLC), the downstream signal molecule of RhoA, was assessed by western blotting. RESULTS: The epithelial barrier disruption and the loss of adherens junctional proteins E-cadherin and ß-catenin in cytomembrane were observed in HDM-treated 16HBE cells, paralleled with the increase of eHsp90α secretion. All of which were rescued in Hsp90α-silencing cells or by pretreating 16HBE cells with 1G6-D7. Also, 1G6-D7 suppressed RhoA activity and MLC phosphorylation induced by HDM. Furthermore, inhibitors of Rho kinase prevented and restored the airway barrier disruption. Consistently, it was hrHsp90α instead of hrHsp90ß that promoted barrier dysfunction and activated RhoA/MLC signaling in 16HBE cells. CONCLUSIONS: The eHsp90α mediates HDM-induced human bronchial epithelial barrier dysfunction by activating RhoA/MLC signaling, suggesting that eHsp90α is a potential therapeutic target for treatment of asthma.

An improved heuristic algorithm for finding motif signals in DNA sequences.

The planted (l, d)-motif search problem is a mathematical abstraction of the DNA functional site discovery task. In this paper, we propose a heuristic algorithm that can find planted (l, d)-signals in a given set of DNA sequences. Evaluations on simulated data sets demonstrate that the proposed algorithm outperforms current widely used motif finding algorithms. We also report the results of experiments on real biological data sets.

[Serum leptin level and its association with bone mineral density in obese children].

OBJECTIVE: To investigate serum leptin level and its relationship with bone mineral density in obese children from Changsha City. METHODS: One hundred and nineteen obese children and 103 normal children aged 7 to 12 years from five primary schools of Changsha City were enrolled. Obesity was assessed based on the body mass index (BMI). Dual energy X-ray absorptiometry (DEXA) was used to determine bone mineral density (BMD) and body composition. Serum leptin level was measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The obesity group had higher height, weight, BMI, waist circumference and waist to hip ratio (WHR) compared with the normal group (p<0.01). BMD, bone mineral content (BMC), lean mass (LM), fat mass (FM), percentage of body fat (%BF) and leptin concentration in the obesity group were significantly higher than those in the normal group (p<0.01). Serum leptin level was positively correlated with BMD, BMC, LM and FM (r=0.528-0.903, p<0.01). Multiple stepwise regression analysis indicated that BMI and %BF were independent influencing factors for serum leptin level. CONCLUSIONS: Obese children have higher serum leptin level. Serum leptin concentration is significantly correlated with BMD and body composition. BMI and %BF are independent influencing factors for serum leptin level in children.

[Plasma levels of adiponectin and tumor necrosis factor-alpha in children with obesity].

OBJECTIVE: To examine plasma adiponectin (ADPN) and tumor necrosis factor-alpha (TNF-alpha) levels and their correlation in children with obesity in order to investigate the roles of both in the development of childhood obesity. METHODS: One hundred and forty-seven children with obesity and 118 normal children who were randomly sampled from five primary schools from the Kaifu District in Changsha were enrolled. Physical shape indexes, including height, weight, waist circumference, hip circumference, and waist to hip ratio (WHR) were measured. Body mass index (BMI) was calculated. Blood pressure was measured. Percentage of body fat (%BF) was measured with dual energy X-ray absorptiometry. Plasmal levels of ADPN and TNF-alpha were detected using ABC-ELISA. Blood concentrations of triglycerides (TG), total cholesterol (TC), high density lipoprotein-cholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) were measured by automatic biochemistry analyzer. Fasting blood glucose level was measured by glucose oxidase method. Fasting blood insulin level was assayed by radioimmunity. Homeostasis model assessment for insulin resistance (HOMA-IR) was performed. RESULTS: Plasma ADPN levels in obese children significantly decreased compared with those in normal children (8.12+/-2.54 mg/L vs 12.22+/-4.68 mg/L; p<0.05), and had a negative correlation with plasma TNF-alpha levels, BMI, WHR and HOMA-IR (p<0.01), and with %BF, fasting insulin, systolic blood pressure and TG (p<0.05). Plasma TNF-alpha levels in obese children significantly increased compared to normal children (171.38+/-34.33 ng/L vs 91.07+/-21.60 ng/L; p<0.01) and positively correlated with BMI, WHR, %BF, fasting insulin, HOMA-IR, TG and systolic blood pressure (p<0.01), and negatively with HDL (p<0.05). Multiple stepwise regression analysis showed that ADPN, BMI and TNF-alpha were main influential factors for %BF (R2=0.926, p<0.01). There was a significant interaction between ADPN and TNF-alpha (p<0.05). CONCLUSIONS: Plasma ADPN levels decreased and plasma TNF-alpha levels increased in children with obesity and both were main influential factors for %BF in children. There was an interaction between ADPN and TNF-alpha, suggesting that they both participate in the development of childhood obesity.

[Obesity and its influencing factors in primary school students from Kaifu District of Changsha City].

OBJECTIVE: To study the prevalence of obesity and the influencing factors for obesity in primary school students from Kaifu District of Changsha City. METHODS: A total of 4 140 students aged 7 to 12 years sampled randomly from Kaifu District of Changsha City were enrolled. Obesity was identified based on the body mass index (BMI). The influencing factors for obesity were investigated by non-logistic regression analysis. RESULTS: The prevalence of overweight and obesity were 9.76% and 7.39% respectively, and the prevalence of obesity in boys and girls were 9.37% and 5.13% respectively (P<0.05). The obese children had significantly higher BMI, waist circumference (WC), waist to hip ratio(WHR), percentage of body fat (%BF), systolic blood pressure, and serum triglyceride (TG) and low density lipoprotein-cholesterol (LDL-C) levels but significantly lower serum high density lipoprotein-cholesterol (HDL-C) level compared with the children with normal weight. BMI, %BF, WC and WHR were all positively correlated to serum levels of TG and LDL-C in obese children. Binge overeating, increased television viewing time, fast speed of eating and increased consumption of fried foods are all risk factors for the development of obesity. Preference for physical activity and sufficient physical activity were protective factors for obesity. CONCLUSIONS: The prevalence of obesity of primary school students from Kaifu District of Changsha City is high in China. The development of childhood obesity is associated with poor eating behaviors, less physical activity and increased television viewing time. The control of childhood obesity may be beneficial to early prevention of some adult chronic diseases.