The rapidly evolving X-linked MIR-506 family fine-tunes spermatogenesis to enhance sperm competition.

Despite rapid evolution across eutherian mammals, the X-linked MIR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes (SLITRK2 and FMR1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked MIR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernible defects, but simultaneous ablation of five clusters containing 19 members of the MIR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility, and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked MIR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the MIR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.

The Rapidly Evolving X-linked miR-506 Family Finetunes Spermatogenesis to Enhance Sperm Competition.

Despite rapid evolution across eutherian mammals, the X-linked miR-506 family miRNAs are located in a region flanked by two highly conserved protein-coding genes (Slitrk2 and Fmr1) on the X chromosome. Intriguingly, these miRNAs are predominantly expressed in the testis, suggesting a potential role in spermatogenesis and male fertility. Here, we report that the X-linked miR-506 family miRNAs were derived from the MER91C DNA transposons. Selective inactivation of individual miRNAs or clusters caused no discernable defects, but simultaneous ablation of five clusters containing nineteen members of the miR-506 family led to reduced male fertility in mice. Despite normal sperm counts, motility and morphology, the KO sperm were less competitive than wild-type sperm when subjected to a polyandrous mating scheme. Transcriptomic and bioinformatic analyses revealed that these X-linked miR-506 family miRNAs, in addition to targeting a set of conserved genes, have more targets that are critical for spermatogenesis and embryonic development during evolution. Our data suggest that the miR-506 family miRNAs function to enhance sperm competitiveness and reproductive fitness of the male by finetuning gene expression during spermatogenesis.

Exploration of whole genome amplification generated chimeric sequences in long-read sequencing data.

MOTIVATION: Multiple displacement amplification (MDA) has become the most commonly used method of whole genome amplification, generating a vast amount of DNA with higher molecular weight and greater genome coverage. Coupling with long-read sequencing, it is possible to sequence the amplicons of over 20 kb in length. However, the formation of chimeric sequences (chimeras, expressed as structural errors in sequencing data) in MDA seriously interferes with the bioinformatics analysis but its influence on long-read sequencing data is unknown. RESULTS: We sequenced the phi29 DNA polymerase-mediated MDA amplicons on the PacBio platform and analyzed chimeras within the generated data. The 3rd-ChimeraMiner has been constructed as a pipeline for recognizing and restoring chimeras into the original structures in long-read sequencing data, improving the efficiency of using TGS data. Five long-read datasets and one high-fidelity long-read dataset with various amplification folds were analyzed. The result reveals that the mis-priming events in amplification are more frequently occurring than widely perceived, and the propor tion gradually accumulates from 42% to over 78% as the amplification continues. In total, 99.92% of recognized chimeric sequences were demonstrated to be artifacts, whose structures were wrongly formed in MDA instead of existing in original genomes. By restoring chimeras to their original structures, the vast majority of supplementary alignments that introduce false-positive structural variants are recycled, removing 97% of inversions on average and contributing to the analysis of structural variation in MDA-amplified samples. The impact of chimeras in long-read sequencing data analysis should be emphasized, and the 3rd-ChimeraMiner can help to quantify and reduce the influence of chimeras. AVAILABILITY AND IMPLEMENTATION: The 3rd-ChimeraMiner is available on GitHub, https://github.com/dulunar/3rdChimeraMiner.

Small RNA shuffling between murine sperm and their cytoplasmic droplets during epididymal maturation.

Reports that mouse sperm gain small RNAs from the epididymosomes secreted by epididymal epithelial cells and that these "foreign" small RNAs act as an epigenetic information carrier mediating the transmission of acquired paternal traits have drawn great attention because the findings suggest that heritable information can flow from soma to germ line, thus invalidating the long-standing Weismann's barrier theory on heritable information flow. Using small RNA sequencing (sRNA-seq), northern blots, sRNA in situ hybridization, and immunofluorescence, we detected substantial changes in the small RNA profile in murine caput epididymal sperm (sperm in the head of the epididymis), and we further determined that the changes resulted from sperm exchanging small RNAs, mainly tsRNAs and rsRNAs, with cytoplasmic droplets rather than the epididymosomes. Moreover, the murine sperm-borne small RNAs were mainly derived from the nuclear small RNAs in late spermatids. Thus, caution is needed regarding sperm gaining foreign small RNAs as an underlying mechanism of epigenetic inheritance.

Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis.

Platelets have emerged as key inflammatory cells implicated in the pathology of sepsis, but their contributions to rapid clinical deterioration and dysregulated inflammation have not been defined. Here, we show that the incidence of thrombocytopathy and inflammatory cytokine release was significantly increased in patients with severe sepsis. Platelet proteomic analysis revealed significant upregulation of gasdermin D (GSDMD). Using platelet-specific Gsdmd-deficient mice, we demonstrated a requirement for GSDMD in triggering platelet pyroptosis in cecal ligation and puncture (CLP)-induced sepsis. GSDMD-dependent platelet pyroptosis was induced by high levels of S100A8/A9 targeting toll-like receptor 4 (TLR4). Pyroptotic platelet-derived oxidized mitochondrial DNA (ox-mtDNA) potentially promoted neutrophil extracellular trap (NET) formation, which contributed to platelet pyroptosis by releasing S100A8/A9, forming a positive feedback loop that led to the excessive release of inflammatory cytokines. Both pharmacological inhibition using Paquinimod and genetic ablation of the S100A8/A9-TLR4 signaling axis improved survival in mice with CLP-induced sepsis by suppressing platelet pyroptosis.

Ablation of the miR-465 Cluster Causes a Skewed Sex Ratio in Mice.

The X-linked miR-465 cluster is highly expressed in the testis, sperm, newborn ovary, and blastocysts as well as in 8-16 cell embryos. However, the physiological role of the miR-465 cluster is still largely unknown. This study aims to dissect the role of the miR-465 cluster in murine development. Despite abundant expression in the testis, ablation of the miR-465 miRNA cluster using CRISPR-Cas9 did not cause infertility. Instead, a skewed sex ratio biased toward males (60% males) was observed among miR-465 KO mice. Further analyses revealed that the female conceptuses selectively degenerated as early as embryonic day 8.5 (E8.5). Small RNA deep sequencing, qPCR, and in situ hybridization analyses revealed that the miRNAs encoded by the miR-465 cluster were mainly localized to the extraembryonic tissue/developing placenta. RNA-seq analyses identified altered mRNA transcriptome characterized by the dysregulation of numerous critical placental genes, e.g., Alkbh1, in the KO conceptuses at E7.5. Taken together, this study showed that the miR-465 cluster is required for normal female placental development, and ablation of the miR-465 cluster leads to a skewed sex ratio with more males (~60%) due to selective degeneration and resorption of the female conceptuses.

[Finite element analysis of different intramedullary fixation methods for the treatment of senile pubic ramus fractures].

OBJECTIVE: To evaluate the biomechanical stability of elastic intramedullary nail in the treatment of pubic ramus fractures by finite element analysis, and to compare the stability of elastic intramedullary nail with cannulated screw intramedullary fixation. METHODS: The CT data of the pelvis of a volunteer were selected, and the three-dimensional model of the pelvis was reconstructed by reverse engineering software and the fracture of the pubic ramus fractures was simulated by osteotomy. The hollow nail model, single elastic nail model and double elastic nailmodel were assembled with different implants respectively. The mesh division, material assignment loading and other steps were carried out in the ANSYS software, and then the calculation was submitted. RESULTS: The overall displacement of the pelvis of the elastic nail model was smaller than that of the cannulated screw model, in which the double elastic nail model had the smallest overall displacement, but the cannulated screw model had the smallest plant displacement and the single elastic nail model had the largest plant displacement. Although the stress of cannulated screw was small, there was obvious stress concentration, the stress of elastic nail was large, but there was no obvious stress concentration, especially the stress distribution of double elastic nail was more uniform and the overall stress of pelvis was the smallest. CONCLUSION: All the three fixation methods can effectively improve the stability of the anterior ring of the pelvis. Among them, there is no significant difference in the overall biomechanical propertiesof hollow nail fixation and double elastic nail fixation, which is better than that of single elastic nail fixation. Elastic nail fixation has the advantages of minimally invasive surgery and good biomechanical stability, so it can be used as a better surgical method for the treatment of pubic ramus fractures.

AQUARIUM: accurate quantification of circular isoforms using model-based strategy.

SUMMARY: Currently, most computational methods estimate the expression of circular RNAs (circRNAs) using the number of sequencing reads that support back-splicing junctions (BSJ) in RNA-seq data, which may introduce biased estimation of circRNA expression due to the uneven distribution of sequencing reads. To overcome this, we previously developed a model-based strategy for circRNA quantification, enabling consideration of sequencing reads from the entire transcript. Yet, the lack of exact transcript structure of circRNAs may limit its accuracy. Here, we proposed a substantially improved circRNA quantification tool, AQUARIUM, by introducing the full-length RNA structure of circular isoforms. We assessed its performance in circRNA quantification using both biological and simulated rRNA-depleted RNA-seq datasets, and demonstrated its superior performance at both BSJ and isoform level. AVAILABILITY AND IMPLEMENTATION: AQUARIUM is freely available at https://github.com/wanjun-group-seu/AQUARIUM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Divergent and Overlapping Roles for Selected Phytochemicals in the Regulation of Pathological Cardiac Hypertrophy.

Plant-based foods, like fruits, vegetables, whole grains, legumes, nuts, seeds and other foodstuffs, have been deemed as heart healthy. The chemicals within these plant-based foods, i.e., phytochemicals, are credited with protecting the heart. However, the mechanistic actions of phytochemicals, which prevent clinical endpoints, such as pathological cardiac hypertrophy, are still being elucidated. We sought to characterize the overlapping and divergent mechanisms by which 18 selected phytochemicals prevent phenylephrine- and phorbol 12-myristate 13-acetate-mediated cardiomyocyte enlargement. Of the tested 18 compounds, six attenuated PE- and PMA-mediated enlargement of neonatal rat ventricular myocytes. Cell viability assays showed that apigenin, baicalein, berberine hydrochloride, emodin, luteolin and quercetin dihydrate did not reduce cell size through cytotoxicity. Four of the six phytochemicals, apigenin, baicalein, berberine hydrochloride and emodin, robustly inhibited stress-induced hypertrophy and were analyzed further against intracellular signaling and genome-wide changes in mRNA expression. The four phytochemicals differentially regulated mitogen-activated protein kinases and protein kinase D. RNA-sequencing further showed divergence in gene regulation, while pathway analysis demonstrated overlap in the regulation of inflammatory pathways. Combined, this study provided a comprehensive analysis of cardioprotective phytochemicals. These data highlight two defining observations: (1) that these compounds predominantly target divergent gene pathways within cardiac myocytes and (2) that regulation of overlapping signaling and gene pathways may be of particular importance for the anti-hypertrophic actions of these phytochemicals. Despite these new findings, future works investigating rodent models of heart failure are still needed to understand the roles for these compounds in the heart.

Peripheral blood non-canonical small non-coding RNAs as novel biomarkers in lung cancer.

One unmet challenge in lung cancer diagnosis is to accurately differentiate lung cancer from other lung diseases with similar clinical symptoms and radiological features, such as pulmonary tuberculosis (TB). To identify reliable biomarkers for lung cancer screening, we leverage the recently discovered non-canonical small non-coding RNAs (i.e., tRNA-derived small RNAs [tsRNAs], rRNA-derived small RNAs [rsRNAs], and YRNA-derived small RNAs [ysRNAs]) in human peripheral blood mononuclear cells and develop a molecular signature composed of distinct ts/rs/ysRNAs (TRY-RNA). Our TRY-RNA signature precisely discriminates between control, lung cancer, and pulmonary TB subjects in both the discovery and validation cohorts and outperforms microRNA-based biomarkers, which bears the diagnostic potential for lung cancer screening.

Clinical Significance of CD147 in Children with Inflammatory Bowel Disease.

BACKGROUND: CD147/basigin (Bsg), a transmembrane glycoprotein, activates matrix metalloproteinases and promotes inflammation. OBJECTIVE: The aim of this study is to explore the clinical significance of CD147 in the pathogenesis of inflammatory bowel disease (IBD). RESULTS: In addition to monocytes, the clinical analysis showed that there is no significance obtained in leucocyte, neutrophil, eosinophil, basophil, and erythrocyte between IBD and controls. Immunohistochemistry analysis showed that CD147 was increased in intestinal tissue of patients with active IBD compared to that in the control group. What is more, CD147 is involved in intestinal barrier function and intestinal inflammation, which was attributed to the fact that it has an influence on MCT4 expression, a regulator of intestinal barrier function and intestinal inflammation, in HT-29 and CaCO2 cells. Most importantly, serum level of CD147 content is higher in active IBD than that in inactive IBD or healthy control, which could be a biomarker of IBD. CONCLUSION: The data suggested that increased CD147 level could be a biomarker of IBD in children.

Clinical Application of Oral Meglumine Diatrizoate Esophagogram in Screening for Esophageal Fistula During Radiotherapy or Chemoradiotherapy for Esophageal Cancer.

Background: This study aimed to investigate the specificity and sensitivity of oral meglumine diatrizoate esophagogram in screening for esophageal fistula during radiotherapy or chemoradiotherapy for esophageal cancer and determine if early detection and intervention could improve the prognosis of esophageal fistulas. Methods: Esophageal cancer patients undergoing radiotherapy or chemoradiotherapy were included. Weekly oral meglumine diatrizoate esophagograms were performed to screen for esophageal fistulas during radiotherapy. When an esophageal fistula was detected, fibroesophagoscopy and computed tomography (CT) were used for confirmation; once confirmed, radiotherapy was discontinued, and the patient received intervention. The esophagogram results were reviewed weekly to assess the recovery of the esophageal fistula. If the fistula was healed, the patient resumed and completed radiotherapy. Results: A total of 206 patients with cancer of the esophagus undergoing chemotherapy/radiotherapy were included. During radiotherapy, 10 cases of esophageal fistula were detected or suspected based on the oral meglumine diatrizoate esophagography findings, and eight of those cases were confirmed by CT and esophagoscopy. All patients with esophageal fistula received intervention; among them, 62.5% (5/8) recovered after 1 to 2 weeks of treatment and continued radiotherapy to completion. The sensitivity and specificity of oral meglumine diatrizoate esophagography in screening for esophageal fistulas during radiotherapy or chemoradiotherapy were 100 and 98.9%, respectively. The median survival period of patients with esophageal fistulas was 6.4 months. Conclusion: Oral meglumine diatrizoate esophagography has high sensitivity and specificity in screening for esophageal fistulas during radiotherapy or chemoradiotherapy with minimal side effects. Early diagnosis and timely intervention can significantly improve the prognosis and prolong the survival period of patients. Trial Registration: Chictr.org.cn, Identifier: ChiCTR-DDD-17012617. Registered on September 7, 2017. The first participant was enrolled on September 25, 2017. http://www.chictr.org.cn/showproj.aspx?proj=21526.

Mast cells-derived MiR-223 destroys intestinal barrier function by inhibition of CLDN8 expression in intestinal epithelial cells.

BACKGROUND: Mast cells (MCs) have been found to play a critical role during development of inflammatory bowel disease (IBD) that characterized by dysregulation of inflammation and impaired intestinal barrier function. However, the function of MCs in IBD remains to be fully elucidated. RESULTS: In our study, we used exosomes isolated from human mast cells-1 (HMCs-1) to culture with NCM460, HT-29 or CaCO2 of intestinal epithelial cells (IECs) to investigate the communication between MCs and IECs. We found that MCs-derived exosomes significantly increased intestinal epithelial permeability and destroyed intestinal barrier function, which is attributed to exosome-mediated functional miRNAs were transferred from HMCs-1 into IECs, leading to inhibit tight junction-related proteins expression, including tight junction proteins 1 (TJP1, ZO-1), Occludin (OCLN), Claudin 8 (CLDN8). Microarray and bioinformatic analysis have further revealed that a panel of miRNAs target different tight junction-related proteins. Interestingly, miR-223 is enriched in mast cell-derived exosome, which inhibit CLDN8 expression in IECs, while treatment with miR-223 inhibitor in HT-29 cells significantly reversed the inhibitory effect of HMCs-1-derived exosomes on CLDN 8 expression. Most importantly, enrichment of MCs accumulation in intestinal mucosa of patients with IBD compared with those healthy control. CONCLUSIONS: These results indicated that enrichment of exosomal miR-223 from HMCs-1 inhibited CLDN8 expression, leading to destroy intestinal barrier function. These finding provided a novel insight of MCs as a new target for therapeutic treatment of IBD.

Mast cells-derived MiR-223 destroys intestinal barrier function by inhibition of CLDN8 expression in intestinal epithelial cells

BACKGROUND: Mast cells (MCs) have been found to play a critical role during development of inflammatory bowel disease (IBD) that characterized by dysregulation of inflammation and impaired intestinal barrier function. However, the function of MCs in IBD remains to be fully elucidated. RESULTS: In our study, we used exosomes isolated from human mast cells-1 (HMCs-1) to culture with NCM460, HT-29 or CaCO2 of intestinal epithelial cells (lECs) to investigate the communication between MCs and lECs. We found that MCs-derived exosomes significantly increased intestinal epithelial permeability and destroyed intestinal barrier function, which is attributed to exosome-mediated functional miRNAs were transferred from HMCs-1 into lECs, leading to inhibit tight junction-related proteins expression, including tight junction proteins 1 (TJP1, ZO-1), Occludin (OCLN), Claudin 8 (CLDN8). Microarray and bioinformatic analysis have further revealed that a panel of miRNAs target different tight junction-related proteins. Interestingly, miR-223 is enriched in mast cell-derived exosome, which inhibit CLDN8 expression in IECs, while treatment with miR-223 inhibitor in HT-29 cells significantly reversed the inhibitory effect of HMCs-1-derived exosomes on CLDN 8 expression. Most importantly, enrichment of MCs accumulation in intestinal mucosa of patients with IBD compared with those healthy control. CONCLUSIONS: These results indicated that enrichment of exosomal miR-223 from HMCs-1 inhibited CLDN8 expression, leading to destroy intestinal barrier function. These finding provided a novel insight of MCs as a new target for therapeutic treatment of IBD.

Inhibition of CREB-mediated ZO-1 and activation of NF-κB-induced IL-6 by colonic epithelial MCT4 destroys intestinal barrier function.

OBJECTIVE: Inflammatory bowel disease (IBD) is a disorder intestinal inflammation and impaired barrier function, associated with increased epithelial expression of monocarboxylate transporter 4 (MCT4). However, the specific non-metabolic function and clinical relevance of MCT4 in IBD remain to be fully elucidated. METHODS: Lentivirus-mediated overexpression of MCT4 was used to assess the role of MCT4 in transcriptionally regulating ZO-1 and IL-6 expression by luciferase assays, WB and ChIP. IP was used to analyse the effect of MCT4 on the interaction NF-κB-CBP or CREB-CBP, and these MCT4-mediated effects were confirmed in vivo assay. RESULTS: We showed that ectopic expression of MCT4 inhibited ZO-1 expression, while increased pro-inflammatory factors expression, leading to destroy intestinal epithelial barrier function in vitro and in vivo. Mechanistically, MCT4 contributed NF-κB p65 nuclear translocation and increased the binding of NF-κB p65 to the promoter of IL-6, which is attributed to MCT4 enhanced NF-κB-CBP interaction and dissolved CREB-CBP complex, resulting in reduction of CREB activity and CREB-mediated ZO-1 expression. In addition, treatment of experimental colitis with MCT4 inhibitor α-cyano-4-hydroxycinnamate (CHC) ameliorated mucosal intestinal barrier function, which was due to attenuation of pro-inflammation factors expression and enhancement of ZO-1 expression. CONCLUSION: These findings suggested a novel role of MCT4 in controlling development of IBD and provided evidence for potential targets of IBD.

Potential Role of HMGCS2 in Tumor Angiogenesis in Colorectal Cancer and Its Potential Use as a Diagnostic Marker.

Objective: HMGCS2 is the rate-limiting enzyme of ketogenesis, which is vital for tumor initiation or metastasis. The aim of this study is to determine the relationship between HMGCS2 and tumor angiogenesis. Materials and Methods: The study consisted of 100 cases with colorectal cancer and healthy control, the expression of HMGCS2 and the microvessel density (MVD) (marker: CD31) were analyzed by immunohistochemistry and tube formation, and the centration of ß-hydroxybutyrate in serum was assessed by biochemical analysis. Results: The results showed that HMGCS2 expression is significantly reduced in colorectal cancer compared with healthy control, which is inversely correlated with MVD in colorectal cancer by IHC analysis. What is more, knockdown HMGCS2 expression in HT-29 cells significantly contributed endothelial cell tube formation. Conclusion: These findings implying HMGCS2 may have a negative regulation of tumor angiogenesis and provide an approach to inhibit tumor angiogenesis.

Immunoglobulin-Like Transcript 5 Inhibits Macrophage-Mediated Bacterial Killing and Antigen Presentation During Sepsis.

BACKGROUND: Immunosuppression contributes to the mortality of sepsis. However, the underlying mechanism remains unclear. METHODS: In the present study, we investigated the role of inhibitory receptor immunoglobulin-like transcript 5 (ILT5) in sepsis. We first screened the expression of ILT family members, and we found that ILT5 was dramatically up-regulated in the peripheral blood mononuclear cells from sepsis patients versus healthy donors. RESULTS: Knockdown of ILT5 by small interfering ribonucleic acid increased bacterial killing and reactive oxygen species production in THP-1 and RAW264.7 cells. Moreover, ILT5-expressing monocytes/macrophages exhibited lower expression of antigen-presenting molecules including major histocompatibility complex-II and CD80. In the in vitro coculture system with monocytes/macrophages, blockage of ILT5 facilitated Th1 proliferation and differentiation of CD4+ T cells. Furthermore, in vivo experiments demonstrated that pretreatment with ILT5 blocking peptide improved the survival and pulmonary pathology of septic mice. CONCLUSIONS: Together, our study identified ILT5 as an immunosuppressive regulator during sepsis, which may provide potential therapeutic strategy for sepsis.

Rat BodyMap transcriptomes reveal unique circular RNA features across tissue types and developmental stages.

Circular RNAs (circRNAs) are a novel class of regulatory RNAs. Here, we present a comprehensive investigation of circRNA expression profiles across 11 tissues and four developmental stages in rats, along with cross-species analyses in humans and mice. Although the expression of circRNAs is positively correlated with that of cognate mRNAs, highly expressed genes tend to splice a larger fraction of circular transcripts. Moreover, circRNAs exhibit higher tissue specificity than cognate mRNAs. Intriguingly, while we observed a monotonic increase of circRNA abundance with age in the rat brain, we further discovered a dynamic, age-dependent pattern of circRNA expression in the testes that is characterized by a dramatic increase with advancing stages of sexual maturity and a decrease with aging. The age-sensitive testicular circRNAs are highly associated with spermatogenesis, independent of cognate mRNA expression. The tissue/age implications of circRNAs suggest that they present unique physiological functions rather than simply occurring as occasional by-products of gene transcription.

Clinical application of oral meglumine diatrizoate esophagogram in screening esophageal fistula during radiotherapy for esophageal cancer.

INTRODUCTION: Esophageal fistula is a serious and common complication of radiotherapy for esophageal cancer. Therefore, early diagnosis and treatment is necessary. Because of side effect of barium esophagography, it cannot be used to screening esophageal fistula during radiotherapy. Meglumine diatrizoate is an ionic contrast agent, its adverse reactions were rarely seen when it was used in the body cavity. The purpose of this trial is identified the sensitivity and specificity of oral meglumine diatrizoate in an esophagogram for screening esophageal fistula during radiotherapy. METHODS/DESIGN: This trial was a prospective, multicenter, diagnostic clinical trial. A total of 105 patients with esophageal cancer will swallowed meglumine diatrizoate and underwent a radiographic examination weekly during radiotherapy, medical personnel observed the esophageal lesions to determine whether an esophageal fistula formed. If an esophageal fistula was observed, esophagofiberoscopy and/or computer tomography was used to further confirm the diagnosis. And the sensitivity and specificity of meglumine diatrizoate should be calculated for screening esophageal fistula during radiotherapy. DISCUSSION: To our knowledge, this study protocol is the first to identify the sensitivity and specificity of oral meglumine diatrizoate in an esophagogram for screening esophageal fistula during radiotherapy. If oral meglumine diatrizoate can be used to screening esophageal fistula, more patients will benefit from early detection and treatment.