Steroid lifting method during endoscopic submucosal dissection: A novel strategy for stricture prevention.

A 73-year-old male patient was referred to us with a long Barrett's esophagus (BE). He had a history of pulmonary embolism under anticoagulant therapy. Esophagogastroduodenoscopy showed a C8M9 BE with no macroscopic lesions. Random biopsies from the BE revealed multifocal high-grade dysplasia. The case was discussed in a multidisciplinary team conference and the decision for full resection of BE with endoscopic submucosal dissection (ESD) was made. Considering the large ESD resection and the high risk of stricture, we developed a novel preventive technique: the "steroid lifting method" for submucosal injection during ESD. Complete circumferential ESD with en bloc resection was performed using the "steroid lifting method", without adverse events. Oral liquids were initiated on day 1 and the patient was discharged on day 4. Oral prednisolone (30 mg per day) was started and tapered for a total of 6 weeks. The pathological examination confirmed multifocal high-grade dysplasia, with radical and curative resection. The patient had neither stricture, dysphagia nor recurrence of Barrett's mucosa at the 2, 6, 12, and 24-month follow-up. International guidelines recommend oral prednisolone and triamcinolone injection to prevent stricture formation in large ESD of esophageal squamous cell carcinoma. However, there is no solid data on BE ESD. The risk factors for stricture formation and the optimal preventive management after large BE ESD is not known. The "steroid lifting method" might be an option in this context. Large prospective studies addressing stricture formation and preventive measures on BE ESD are necessary.

A landscape of gene expression regulation for synovium in arthritis.

The synovium is an important component of any synovial joint and is the major target tissue of inflammatory arthritis. However, the multi-omics landscape of synovium required for functional inference is absent from large-scale resources. Here we integrate genomics with transcriptomics and chromatin accessibility features of human synovium in up to 245 arthritic patients, to characterize the landscape of genetic regulation on gene expression and the regulatory mechanisms mediating arthritic diseases predisposition. We identify 4765 independent primary and 616 secondary cis-expression quantitative trait loci (cis-eQTLs) in the synovium and find that the eQTLs with multiple independent signals have stronger effects and heritability than single independent eQTLs. Integration of genome-wide association studies (GWASs) and eQTLs identifies 84 arthritis related genes, revealing 38 novel genes which have not been reported by previous studies using eQTL data from the GTEx project or immune cells. We further develop a method called eQTac to identify variants that could affect gene expression by affecting chromatin accessibility and identify 1517 regions with potential regulatory function of chromatin accessibility. Altogether, our study provides a comprehensive synovium multi-omics resource for arthritic diseases and gains new insights into the regulation of gene expression.

Topflight endoscopic submucosal dissection: a novel strategy for the resection of gastric fundus tumors.

Video 1Resection of a gastric lesion using Topflight ESD.

Enhancer variants on chromosome 2p14 regulating SPRED2 and ACTR2 act as a signal amplifier to protect against rheumatoid arthritis.

Genome-wide association studies (GWASs) have repeatedly reported multiple non-coding single-nucleotide polymorphisms (SNPs) at 2p14 associated with rheumatoid arthritis (RA), but their functional roles in the pathological mechanisms of RA remain to be explored. In this study, we integrated a series of bioinformatics and functional experiments and identified three intronic RA SNPs (rs1876518, rs268131, and rs2576923) within active enhancers that can regulate the expression of SPRED2 directly. At the same time, SPRED2 and ACTR2 influence each other as a positive feedback signal amplifier to strengthen the protective role in RA by inhibiting the migration and invasion of rheumatoid fibroblast-like synoviocytes (FLSs). In particular, the transcription factor CEBPB preferentially binds to the rs1876518-T allele to increase the expression of SPRED2 in FLSs. Our findings decipher the molecular mechanisms behind the GWAS signals at 2p14 for RA and emphasize SPRED2 as a potential candidate gene for RA, providing a potential target and direction for precise treatment of RA.

Duodenal subepithelial neuroendocrine tumor removed by endoscopic submucosal dissection using internal traction with magnets.

Video 1Endoscopic submucosal dissection of a duodenal subepithelial neuroendocrine tumor using internal traction with magnets.

Individualized pathway activity algorithm identifies oncogenic pathways in pan-cancer analysis.

BACKGROUND: Accumulative evidences have shown that dysregulation of biological pathways contributed to the initiation and progression of malignant tumours. Several methods for pathway activity measurement have been proposed, but they are restricted to making comparisons between groups or sensitive to experimental batch effects. METHODS: We introduced a novel method for individualized pathway activity measurement (IPAM) that is based on the ranking of gene expression levels in individual sample. Taking advantage of IPAM, we calculated the pathway activity of 318 pathways from KEGG database in the 10528 tumour/normal samples of 33 cancer types from TCGA to identify characteristic dysregulated pathways among different cancer types. FINDINGS: IPAM precisely quantified the level of activity of each pathway in pan-cancer analysis and exhibited better performance in cancer classification and prognosis prediction over five widely used tools. The average ROC-AUC of cancer diagnostic model using tumour-educated platelets (TEPs) reached 92.84%, suggesting the potential of our algorithm in early diagnosis of cancer. We identified several pathways significantly deregulated and associated with patient survival in a large fraction of cancer types, such as tyrosine metabolism, fatty acid degradation, cell cycle, p53 signalling pathway and DNA replication. We also confirmed the dominant role of metabolic pathways in cancer pathway dysregulation and identified the driving factors of specific pathway dysregulation, such as PPARA for branched-chain amino acid metabolism and NR1I2, NR1I3 for fatty acid metabolism. INTERPRETATION: Our study will provide novel clues for understanding the pathological mechanisms of cancer, ultimately paving the way for personalized medicine of cancer. FUNDING: A full list of funding can be found in the Acknowledgements section.

Transcriptome-wide association study identifies multiple genes and pathways associated with thyroid function.

Thyroid dysfunction is a common endocrine disease measured by thyroid-stimulating hormone (TSH) level. Although >70 genetic loci associated with TSH have been reported through genome-wide association studies (GWASs), the variants can only explain a small fraction of the thyroid function heritability. To identify novel candidate genes for thyroid function, we conducted the first large-scale transcriptome-wide association study (TWAS) for thyroid function using GWAS-summary data for TSH levels in up to 119 715 individuals combined with precomputed gene expression weights of six panels from four tissue types. The candidate genes identified by TWAS were further validated by TWAS replication and gene expression profiles. We identified 74 conditionally independent genes significantly associated with thyroid function, such as PDE8B (P = 1.67 × 10-282), PDE10A (P = 7.61 × 10-119), NR3C2 (P = 1.50 × 10-92) and CAPZB (P = 3.13 × 10-79). After TWAS replication using UKBB datasets, 26 genes were replicated for significant associations with thyroid-relevant diseases/traits. Among them, 16 genes were causal for their associations to thyroid-relevant diseases/traits and further validated in differential expression analyses, including two novel genes (MFSD6 and RBM47) that did not implicate in previous GWASs. Enrichment analyses detected several pathways associated with thyroid function, such as the cAMP signaling pathway (P = 7.27 × 10-4), hemostasis (P = 3.74 × 10-4), and platelet activation, signaling and aggregation (P = 9.98 × 10-4). Our study identified multiple candidate genes and pathways associated with thyroid function, providing novel clues for revealing the genetic mechanisms of thyroid function and disease.

Endoscopic submucosal dissection by using a new traction device.

Video 1Patient with a history of gastric ectopic pancreas and epigastric pain. We illustrate the endoscopic submucosal dissection of the ectopic pancreas using a new traction device, the ProdiGi traction wire. Using this device, we were able to resect the lesion en bloc with no adverse events.

Wide-field endoscopic submucosal dissection for the treatment of Barrett's esophagus neoplasia.

Background and study aims Implementation of endoscopic submucosal dissection (ESD) for the treatment of Barrett's esophagus neoplasia (BEN) has been hampered by high rates of positive margins and complications. Dissection with wider margins was proposed to overcome these problems, but was never tested. We aim to compare Wide-Field ESD (WF-ESD) with conventional ESD (C-ESD) for treatment of BEN. Patients and methods This was a cohort study of all ESDs performed in our center during 2011 to 2018. C-ESD was the only technique used before 2014, with WF-ESD used beginning in 2014. In WF-ESD marking was performed 10 mm from the tumor margin compared to 5 mm with C-E. Results ESD was performed in 90 cases, corresponding to 74 patients, 84 % male, median age 69. Of these, 22 were C-ESD (24 %) and 68 were WF-ESD (76 %). The en bloc resection rate was 95 vs 100 % (ns), the positive lateral margin rate was 23 % vs 3 % ( P  < 0.01), the R0 rate was 73 % vs 90 %, and the curative resection rate was 59 % vs 76 % in the C-ESD and WF-ESD groups, respectively, (both P  > 0.05). The procedure speed was 4.4 and 2.3 (min/mm) in the C-ESD and WF-ESD groups ( P  < 0.01), respectively. WF-ESD was associated with less post-operative strictures, 6 % vs 27 % ( P  = 0.01), with no local recurrence but no significantly reduced risk of metachronous recurrence (Hazard Ratio = 0.46, 95 %CI = 0.14-1.46), during a follow-up of 13.4 and 9.4 months in the C-ESD and WF-ESD cohorts, respectively. Conclusions WF-ESD is associated with a reduction in positive lateral margins, faster dissection, and lower stricture rates. Further prospective, multicenter studies are warranted to evaluate its role in clinical practice.

Modeling circRNA expression pattern with integrated sequence and epigenetic features demonstrates the potential involvement of H3K79me2 in circRNA expression.

MOTIVATION: CircRNAs are an abundant class of non-coding RNAs with widespread, cell-/tissue-specific patterns. Previous work suggested that epigenetic features might be related to circRNA expression. However, the contribution of epigenetic changes to circRNA expression has not been investigated systematically. Here, we built a machine learning framework named CIRCScan, to predict circRNA expression in various cell lines based on the sequence and epigenetic features. RESULTS: The predicted accuracy of the expression status models was high with area under the curve of receiver operating characteristic (ROC) values of 0.89-0.92 and the false-positive rates of 0.17-0.25. Predicted expressed circRNAs were further validated by RNA-seq data. The performance of expression-level prediction models was also good with normalized root-mean-square errors of 0.28-0.30 and Pearson's correlation coefficient r over 0.4 in all cell lines, along with Spearman's correlation coefficient ρ of 0.33-0.46. Noteworthy, H3K79me2 was highly ranked in modeling both circRNA expression status and levels across different cells. Further analysis in additional nine cell lines demonstrated a significant enrichment of H3K79me2 in circRNA flanking intron regions, supporting the potential involvement of H3K79me2 in circRNA expression regulation. AVAILABILITY AND IMPLEMENTATION: The CIRCScan assembler is freely available online for academic use at https://github.com/johnlcd/CIRCScan. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An Allele-Specific Functional SNP Associated with Two Systemic Autoimmune Diseases Modulates IRF5 Expression by Long-Range Chromatin Loop Formation.

Both systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) are autoimmune diseases sharing similar genetic backgrounds. Genome-wide association studies have constantly disclosed numerous genetic variants conferring to both disease risks at 7q32.1, but the functional mechanisms underlying them are still largely unknown. Through a series of bioinformatics and functional analyses, we prioritized a potential independent functional single-nucleotide polymorphism (rs13239597) within TNPO3 promoter region, residing in a putative enhancer element and validated that IRF5 is the distal target gene (∼118 kb) of rs13239597, which is a key regulator involved in pathogenic autoantibody dysregulation, increasing risk of both SLE and SSc. We experimentally validated the long-range chromatin interactions between rs13239597 and IRF5 using chromosome conformation capture assay. We further demonstrated that rs13239597-A acted as an allele-specific enhancer regulating IRF5 expression, independently of TNPO3 by using dual-luciferase reporter assays and CRISPR-Cas9. Particularly, the transcription factor EVI1 could preferentially bind to rs13239597-A allele and increase the enhancer activity to regulate IRF5 expression. Taken together, our results uncovered a mechanistic insight of a noncoding functional variant acting as an allele-specific distal enhancer to directly modulate IRF5 expression, which might obligate in understanding of complex genetic architectures of SLE and SSc pathogenesis.

Unexpected Role of Matrix Gla Protein in Osteoclasts: Inhibiting Osteoclast Differentiation and Bone Resorption.

Matrix Gla protein (MGP) is an extracellular protein responsible for inhibiting mineralization. MGP inhibits osteoblast mineralization and bone formation by regulating the deposition of bone matrix. However, Mgp-/- mice display an osteopenic phenotype. To explain this contradiction, we investigated the role of MGP in osteoclastogenesis, the other side of bone remodeling. We found that MGP expression is markedly increased by osteoclastic commitment. Osteoclast differentiation and bone resorption are accelerated by MGP depletion while suppressed by MGP overexpression. The in vivo results confirmed its inhibitory role in osteoclastogenesis by the administration of Cre-dependent FLEX-On recombinant MGP-AAV to LysM Cre mice. Furthermore, we found that the expression and nuclear translocation of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), are under the control of MGP. MGP loss results in elevation of intracellular Ca2+ flux. Vitronectin-induced activation of Src/Rac1 is magnified in the absence of MGP but reduced when MGP is overexpressed. Inhibition of Src activation or NFATc1 nuclear import rescues the increased osteoclastogenesis induced by MGP deficiency. These observations (i) establish, for the first time to our knowledge, that MGP plays an essential role in osteoclast differentiation and function, (ii) enrich the current knowledge of MGP function, and (iii) indicate the potential of MGP as a therapeutic target for low-bone-mass disorders.

Multiple Functional Variants at 13q14 Risk Locus for Osteoporosis Regulate RANKL Expression Through Long-Range Super-Enhancer.

RANKL is a key regulator involved in bone metabolism, and a drug target for osteoporosis. The clinical diagnosis and assessment of osteoporosis are mainly based on bone mineral density (BMD). Previous powerful genomewide association studies (GWASs) have identified multiple intergenic single-nucleotide polymorphisms (SNPs) located over 100 kb upstream of RANKL and 65 kb downstream of AKAP11 at 13q14.11 for osteoporosis. Whether these SNPs exert their roles on osteoporosis through RANKL is unknown. In this study, we conducted integrative analyses combining expression quantitative trait locus (eQTL), genomic chromatin interaction (high-throughput chromosome conformation capture [Hi-C]), epigenetic annotation, and a series of functional assays. The eQTL analysis identified six potential functional SNPs (rs9533090, rs9594738, r8001611, rs9533094, rs9533095, and rs9594759) exclusively correlated with RANKL gene expression (p < 0.001) at 13q14.11. Co-localization analyses suggested that eQTL signal for RANKL and BMD-GWAS signal shared the same causal variants. Hi-C analysis and functional annotation further validated that the first five osteoporosis SNPs are located in a super-enhancer region to regulate the expression of RANKL via long-range chromosomal interaction. Particularly, dual-luciferase assay showed that the region harboring rs9533090 in the super-enhancer has the strongest enhancer activity, and rs9533090 is an allele-specific regulatory SNP. Furthermore, deletion of the region harboring rs9533090 using CRISPR/Cas9 genome editing significantly reduced RANKL expression in both mRNA level and protein level. Finally, we found that the rs9533090-C robustly recruits transcription factor NFIC, which efficiently elevates the enhancer activity and increases the RANKL expression. In summary, we provided a feasible method to identify regulatory noncoding SNPs to distally regulate their target gene underlying the pathogenesis of osteoporosis by using bioinformatics data analyses and experimental validation. Our findings would be a potential and promising therapeutic target for precision medicine in osteoporosis. © 2018 American Society for Bone and Mineral Research.

The proliferation marker thymidine kinase 1 level is high in normal kidney tubule cells compared to other normal and malignant renal cells.

The activity of the proliferation related enzyme thymidine kinase 1 (TK1) was reported to be 3-fold higher in extracts from normal kidney tissue as compare to renal carcinoma extracts [3]. To verify these unexpected results, determinations of the protein levels of TK1 in normal kidney and in samples from different types of renal cell carcinoma (RCC) were done with immunohistochemistry and Western blot analysis. Two anti-TK1 peptide antibodies reacting with different TK1 epitops were used. TK1 levels were high in tubule cells as compared to glomerulus cells and connective tissue cells, while an intermediary TK1 was observed in renal cell carcinoma (RCC) cells. Western blot analysis demonstrated high levels of TK1 in extract from normal kidney, and lower levels of TK1 in the RCC extracts. The specificity of TK1 staining was demonstrated in competition experiments with excess TK1 antigen. The high TK1 levels in normal kidney tubule cells suggest that they are in a form of activated G1-state. The relatively low TK1 level in RCC, representing TK1 expression in S-phase cells, is in accordance with the low overall proliferation rate of these tumors. These results suggest that cell cycle regulation of TK1 in normal tubule cells differ from that in other type of normal and malignant renal cells.

Thymidine kinase 1 expression defines an activated G1 state of the cell cycle as revealed with site-specific antibodies and ArrayScan assays.

Thymidine kinase 1 (TK1) is a DNA salvage enzyme involved in the synthesis of thymidine triphosphate needed during S phase. Although TK1 has been utilized as a cell proliferation marker for many years no well-characterized antibodies are available. The preparation and properties of two types of poly- and monoclonal anti-TK1 peptide antibodies are described and they are used to determine the levels of TK1 in intact cells. Expression of TK1, c-fos, cyclin B1, Ki67, phosphorylated histone H3, phosphorylated ribosomal protein S6, as well as bromodeoxyuridine (BrdU) incorporation in human normal dermal fibroblast cultures were studied with high-content ArrayScan fluorescence microscopy. The levels of TK1 increased 6-7h after serum re-addition to starved cells as they passed through G1, S and G2/M phases, which was earlier than the increase in Ki67 protein levels and before BrdU incorporation was detected. Thus, a population of activated G1 cells with high TK1 and low Ki67 expression could be identified and their role in cell proliferation can now be clarified.

Cytosolic thymidine kinase is a specific histopathologic tumour marker for breast carcinomas.

Thymidine kinase 1 (TK1), an enzyme involved in the synthesis of precursors for DNA, and thus proliferation dependent, has been suggested as a good tumour marker. We have recently developed poly/monoclonal antibodies against TK1, which proved useful for diagnostics in both serum and immunohistochemistry of cancer patients. The anti-TK1 monoclonal antibodies (mAbs) 1D11 and 1E3 were characterized by Western blot, immunoprecipitation and flow cytometry. TK1 mAbs and Ki-67 mAb were then used for immunohistochemistry staining of tumour sections from 54 patients with ductal infiltrated breast carcinoma. Results showed the relative number of patients with positively stained tumours for TK1 (mAb 1D11) and for Ki-67 (mAb MIB-1) were 47 and 41%, respectively, significantly related (p=0.007). Combination of TK1 mAbs 1D11 and 1E3 increased this number to 56%, due to detection of a significantly higher number of patients with grade 2 tumours. Patients with stage II and grade 2 tumours showed significantly higher TK1 staining when compared to stage I and grade 1. Ki-67 staining was significantly higher in stage III and grade 3. The tumours only stained for TK1 represented higher stages and grades, while tumours staining only for Ki-67 were of lower stages and grades. Combining TK1 and Ki-67 increased the number of patients with positively stained tumours to 69%. In conclusion, TK1 is a reliable marker for identification of patients with grade 2 tumours. The highest number of patients with positively stained tumours were obtained when both TK1 and Ki-67 markers were used.