Integrative Single Cell Atlas Revealed Intratumoral Heterogeneity Generation from an Adaptive Epigenetic Cell State in Human Bladder Urothelial Carcinoma.

Intratumor heterogeneity (ITH) of bladder cancer (BLCA) contributes to therapy resistance and immune evasion affecting clinical prognosis. The molecular and cellular mechanisms contributing to BLCA ITH generation remain elusive. It is found that a TM4SF1-positive cancer subpopulation (TPCS) can generate ITH in BLCA, evidenced by integrative single cell atlas analysis. Extensive profiling of the epigenome and transcriptome of all stages of BLCA revealed their evolutionary trajectories. Distinct ancestor cells gave rise to low-grade noninvasive and high-grade invasive BLCA. Epigenome reprograming led to transcriptional heterogeneity in BLCA. During early oncogenesis, epithelial-to-mesenchymal transition generated TPCS. TPCS has stem-cell-like properties and exhibited transcriptional plasticity, priming the development of transcriptionally heterogeneous descendent cell lineages. Moreover, TPCS prevalence in tumor is associated with advanced stage cancer and poor prognosis. The results of this study suggested that bladder cancer interacts with its environment by acquiring a stem cell-like epigenomic landscape, which might generate ITH without additional genetic diversification.

Non-invasive diagnosis and surveillance of bladder cancer with driver and passenger DNA methylation in a prospective cohort study.

BACKGROUND: State-of-art non-invasive diagnosis processes for bladder cancer (BLCA) harbour shortcomings such as low sensitivity and specificity, unable to distinguish between high- (HG) and low-grade (LG) tumours, as well as inability to differentiate muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC). This study investigates a comprehensive characterization of the entire DNA methylation (DNAm) landscape of BLCA to determine the relevant biomarkers for the non-invasive diagnosis of BLCA. METHODS: A total of 304 samples from 224 donors were enrolled in this multi-centre, prospective cohort study. BLCA-specific DNAm signature discovery was carried out with genome-wide bisulfite sequencing in 32 tumour tissues and 12 normal urine samples. A targeted sequencing assay for BLCA-specific DNAm signatures was developed to categorize tumour tissue against normal urine, or MIBC against NMIBC. Independent validation was performed with targeted sequencing of 259 urine samples in a double-blinded manner to determine the clinical diagnosis and prognosis value of DNAm-based classification models. Functions of genomic region harbouring BLCA-specific DNAm signature were validated with biological assays. Concordances of pathology to urine tumour DNA (circulating tumour DNA [ctDNA]) methylation, genomic mutations or other state-of-the-art diagnosis methods were measured. RESULTS: Genome-wide DNAm profile could accurately classify LG tumour from HG tumour (LG NMIBC vs. HG NMIBC: p = .038; LG NMIBC vs. HG MIBC, p = .00032; HG NMIBC vs. HG MIBC: p = .82; Student's t-test). Overall, the DNAm profile distinguishes MIBC from NMIBC and normal urine. Targeted-sequencing-based DNAm signature classifiers accurately classify LG NMIBC tissues from HG MIBC and could detect tumours in urine at a limit of detection of less than .5%. In tumour tissues, DNAm accurately classifies pathology, thus outperforming genomic mutation or RNA expression profiles. In the independent validation cohort, pre-surgery urine ctDNA methylation outperforms fluorescence in situ hybridization (FISH) assay to detect HG BLCA (n = 54) with 100% sensitivity (95% CI: 82.5%-100%) and LG BLCA (n = 26) with 62% sensitivity (95% CI: 51.3%-72.7%), both at 100% specificity (non-BLCA: n = 72; 95% CI: 84.1%-100%). Pre-surgery urine ctDNA methylation signature correlates with pathology and predicts recurrence and metastasis. Post-surgery urine ctDNA methylation (n = 61) accurately predicts recurrence-free survival within 180 days, with 100% accuracy. CONCLUSION: With the discovery of BLCA-specific DNAm signatures, targeted sequencing of ctDNA methylation outperforms FISH and DNA mutation to detect tumours, predict recurrence and make prognoses.

Species Diversity and Chemotypes of Fusarium Species Associated With Maize Stalk Rot in Yunnan Province of Southwest China.

Maize stalk rot caused by Fusarium species is one of the most important fungal diseases of maize throughout the world. The disease is responsible for considerable yield losses and has also been associated with mycotoxin contamination of the crop. In this study, a survey of maize stalk rot was performed in seven locations of Yunnan Province in China during the cropping season of 2015 and 2016. Based on morphological and molecular characteristics, 204 isolates belonging to 12 Fusarium spp. from symptomatic stalks of maize were identified. Among the isolated strains, 83 were identified as Fusarium meridionale (40.5%), 46 as Fusarium boothii (22.5%), 34 as Fusarium temperatum (16.5%), 12 as Fusarium equiseti (5.9%), 10 as Fusarium asiaticum (4.9%), six as Fusarium proliferatum (3.0%), four as Fusarium verticillioides (2.0%), four as Fusarium incarnatum (2.0%), two as Fusarium avenaceum (1.0%), one as Fusarium cerealis (0.5%), one as Fusarium graminearum (0.5%), and one as Fusarium cortaderiae (0.5%). Fusarium cortaderiae was the first report on the causal agent of maize stalk rot disease in China. These isolates were divided into five chemotypes: nivalenol (NIV), deoxynivalenol (DON), beauvericin (BEA), zearalenone (ZEN), and fumonisin (FUM). Phylogenetic analysis based on partial sequences of the translation elongation factor 1α (TEF1-α) showed a high degree of interspecific polymorphisms among the isolates. Pathogenicity analysis on maize stalks indicated that all the 12 species of Fusarium were able to cause the disease symptoms with different aggressiveness. This study on population, pathogenicity, and toxigenic chemotypes of Fusarium species associated with maize stalk rot in Yunnan Province of southwest China, will help design an effective integrated control strategy for this disease.

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of toxigenic Fusarium temperatum in maize stalks and kernels.

Fusarium temperatum is an emerging maize pathogen that causes maize ear and stalk rot diseases and produces various mycotoxins including moniliformin, beauvericin, enniatins and fumonisin B1, which poses a potential risk to the human food or animal feed supply chains. Early detection of F. temperatum is crucial to prevent its derived mycotoxins from entering the food chain, and is also a useful tool in disease management practices. Here, we describe a loop-mediated isothermal amplification (LAMP) assay for rapid diagnosis of F. temperatum. The 28S ribosomal DNA sequences (28S rDNA) of F. temperatum were used to design a set of six primers. The reaction conditions were optimized for developing a fast assay with high specificity and sensitivity, and were able to detect the presence of less than 10 pg of target DNA per reaction within 60 min. Furthermore, the resulting amplicons were visualized by adding SYBR Green I to the reaction tubes. Suspected F. temperatum infected maize stalk samples collected from Yunnan province, China were identified using the developed LAMP assay. In conclusion, the method not only provides a rapid and specific screening for the existence of F. temperatum in a bulk of maize samples without using sophisticated equipment, but also is potentially useful for other agriculturally important toxigenic fungi.