The EN-TEx resource of multi-tissue personal epigenomes & variant-impact models.

Understanding how genetic variants impact molecular phenotypes is a key goal of functional genomics, currently hindered by reliance on a single haploid reference genome. Here, we present the EN-TEx resource of 1,635 open-access datasets from four donors (∼30 tissues × âˆ¼15 assays). The datasets are mapped to matched, diploid genomes with long-read phasing and structural variants, instantiating a catalog of >1 million allele-specific loci. These loci exhibit coordinated activity along haplotypes and are less conserved than corresponding, non-allele-specific ones. Surprisingly, a deep-learning transformer model can predict the allele-specific activity based only on local nucleotide-sequence context, highlighting the importance of transcription-factor-binding motifs particularly sensitive to variants. Furthermore, combining EN-TEx with existing genome annotations reveals strong associations between allele-specific and GWAS loci. It also enables models for transferring known eQTLs to difficult-to-profile tissues (e.g., from skin to heart). Overall, EN-TEx provides rich data and generalizable models for more accurate personal functional genomics.

Less-is-more: selecting transcription factor binding regions informative for motif inference.

Numerous statistical methods have emerged for inferring DNA motifs for transcription factors (TFs) from genomic regions. However, the process of selecting informative regions for motif inference remains understudied. Current approaches select regions with strong ChIP-seq signal for a given TF, assuming that such strong signal primarily results from specific interactions between the TF and its motif. Additionally, these selection approaches do not account for non-target motifs, i.e. motifs of other TFs; they presume the occurrence of these non-target motifs infrequent compared to that of the target motif, and thus assume these have minimal interference with the identification of the target. Leveraging extensive ChIP-seq datasets, we introduced the concept of TF signal 'crowdedness', referred to as C-score, for each genomic region. The C-score helps in highlighting TF signals arising from non-specific interactions. Moreover, by considering the C-score (and adjusting for the length of genomic regions), we can effectively mitigate interference of non-target motifs. Using these tools, we find that in many instances, strong ChIP-seq signal stems mainly from non-specific interactions, and the occurrence of non-target motifs significantly impacts the accurate inference of the target motif. Prioritizing genomic regions with reduced crowdedness and short length markedly improves motif inference. This 'less-is-more' effect suggests that ChIP-seq region selection warrants more attention.

RNA m6A detection using raw current signals and basecalling errors from Nanopore direct RNA sequencing reads.

MOTIVATION: Nanopore direct RNA sequencing (DRS) enables the detection of RNA N6-methyladenosine (m6A) without extra laboratory techniques. A number of supervised or comparative approaches have been developed to identify m6A from Nanopore DRS reads. However, existing methods typically utilize either statistical features of the current signals or basecalling-error features, ignoring the richer information of the raw signals of DRS reads. RESULTS: Here, we propose RedNano, a deep-learning method designed to detect m6A from Nanopore DRS reads by utilizing both raw signals and basecalling errors. RedNano processes the raw-signal feature and basecalling-error feature through residual networks. We validated the effectiveness of RedNano using synthesized, Arabidopsis, and human DRS data. The results demonstrate that RedNano surpasses existing methods by achieving higher area under the ROC curve (AUC) and area under the precision-recall curve (AUPRs) in all three datasets. Furthermore, RedNano performs better in cross-species validation, demonstrating its robustness. Additionally, when detecting m6A from an independent dataset of Populus trichocarpa, RedNano achieves the highest AUC and AUPR, which are 3.8%-9.9% and 5.5%-13.8% higher than other methods, respectively. AVAILABILITY AND IMPLEMENTATION: The source code of RedNano is freely available at https://github.com/Derryxu/RedNano.

Building integrative functional maps of gene regulation.

Every cell in the human body inherits a copy of the same genetic information. The three billion base pairs of DNA in the human genome, and the roughly 50 000 coding and non-coding genes they contain, must thus encode all the complexity of human development and cell and tissue type diversity. Differences in gene regulation, or the modulation of gene expression, enable individual cells to interpret the genome differently to carry out their specific functions. Here we discuss recent and ongoing efforts to build gene regulatory maps, which aim to characterize the regulatory roles of all sequences in a genome. Many researchers and consortia have identified such regulatory elements using functional assays and evolutionary analyses; we discuss the results, strengths and shortcomings of their approaches. We also discuss new techniques the field can leverage and emerging challenges it will face while striving to build gene regulatory maps of ever-increasing resolution and comprehensiveness.

To mock or not: a comprehensive comparison of mock IP and DNA input for ChIP-seq.

Chromatin immunoprecipitation (IP) followed by sequencing (ChIP-seq) is the gold standard to detect transcription-factor (TF) binding sites in the genome. Its success depends on appropriate controls removing systematic biases. The predominantly used controls, i.e. DNA input, correct for uneven sonication, but not for nonspecific interactions of the IP antibody. Another type of controls, 'mock' IP, corrects for both of the issues, but is not widely used because it is considered susceptible to technical noise. The tradeoff between the two control types has not been investigated systematically. Therefore, we generated comparable DNA input and mock IP experiments. Because mock IPs contain only nonspecific interactions, the sites predicted from them using DNA input indicate the spurious-site abundance. This abundance is highly correlated with the 'genomic activity' (e.g. chromatin openness). In particular, compared to cell lines, complex samples such as whole organisms have more spurious sites-probably because they contain multiple cell types, resulting in more expressed genes and more open chromatin. Consequently, DNA input and mock IP controls performed similarly for cell lines, whereas for complex samples, mock IP substantially reduced the number of spurious sites. However, DNA input is still informative; thus, we developed a simple framework integrating both controls, improving binding site detection.

S100A4 Promotes BCG-Induced Pyroptosis of Macrophages by Activating the NF-κB/NLRP3 Inflammasome Signaling Pathway.

Pyroptosis is a host immune strategy to defend against Mycobacterium tuberculosis (Mtb) infection. S100A4, a calcium-binding protein that plays an important role in promoting cancer progression as well as the pathophysiological development of various non-tumor diseases, has not been explored in Mtb-infected hosts. In this study, transcriptome analysis of the peripheral blood of patients with pulmonary tuberculosis (PTB) revealed that S100A4 and GSDMD were significantly up-regulated in PTB patients' peripheral blood. Furthermore, there was a positive correlation between the expression of GSDMD and S100A4. KEGG pathway enrichment analysis showed that differentially expressed genes between PTB patients and healthy controls were significantly related to inflammation, such as the NOD-like receptor signaling pathway and NF-κB signaling pathway. To investigate the regulatory effects of S100A4 on macrophage pyroptosis, THP-1 macrophages infected with Bacillus Calmette-Guérin (BCG) were pre-treated with exogenous S100A4, S100A4 inhibitor or si-S100A4. This research study has shown that S100A4 promotes the pyroptosis of THP-1 macrophages caused by BCG infection and activates NLRP3 inflammasome and NF-κB signaling pathways, which can be inhibited by knockdown or inhibition of S100A4. In addition, inhibition of NF-κB or NLRP3 blocks the promotion effect of S100A4 on BCG-induced pyroptosis of THP-1 macrophages. In conclusion, S100A4 activates the NF-κB/NLRP3 inflammasome signaling pathway to promote macrophage pyroptosis induced by Mtb infection. These data provide new insights into how S100A4 affects Mtb-induced macrophage pyroptosis.

Endoplasmic Reticulum Stress Mediated NLRP3 Inflammasome Activation and Pyroptosis in THP-1 Macrophages Infected with Bacillus Calmette-Guérin.

Tuberculosis (TB) is a zoonotic infectious disease caused by Mycobacterium tuberculosis (Mtb). Mtb is a typical intracellular parasite, and macrophages are its main host cells. NLRP3 inflammasome-mediated pyroptosis is a form of programmed cell death implicated in the clearance of pathogenic infections. The bidirectional regulatory effect of endoplasmic reticulum stress (ERS) plays a crucial role in determining cell survival and death. Whether ERS is involved in macrophage pyroptosis with Mtb infection remains unclear. This article aims to explore the regulation of the NLRP3 inflammasome and pyroptosis by ERS in THP-1 macrophages infected with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). The results showed that BCG infection induced THP-1 macrophage ERS, NLRP3 inflammasome activation and pyroptosis, which was inhibited by ERS inhibitor TUDCA. NLRP3 inhibitor MCC950 inhibited THP-1 macrophage NLRP3 inflammasome activation and pyroptosis caused by BCG infection. Compared with specific Caspase-1 inhibitor VX-765, pan-Caspase inhibitor Z-VAD-FMK showed a more significant inhibitory effect on BCG infection-induced pyroptosis of THP-1 macrophages. Taken together, this study demonstrates that ERS mediated NLRP3 inflammasome activation and pyroptosis after BCG infection of THP-1 macrophages, and that BCG infection of THP-1 macrophages induces pyroptosis through canonical and noncanonical pathways.

Correction: Nie et al. Endoplasmic Reticulum Stress Mediated NLRP3 Inflammasome Activation and Pyroptosis in THP-1 Macrophages Infected with Bacillus Calmette-Guérin. Int. J. Mol. Sci. 2023, 24, 11692.

Lei Liu was not included as an author in the original publication [...].

GSK2656157, a PERK Inhibitor, Alleviates Pyroptosis of Macrophages Induced by Mycobacterium Bacillus Calmette-Guerin Infection.

Tuberculosis (TB) is the leading cause of human death worldwide due to Mycobacterium tuberculosis (Mtb) infection. Mtb infection can cause macrophage pyroptosis. PERK, as a signaling pathway protein on the endoplasmic reticulum, plays an important role in infectious diseases. It is not clear whether PERK is involved in the regulation of pyroptosis of macrophages during Mtb infection. In this study, Bacillus Calmette-Guerin (BCG) infection resulted in high expression of pro-caspase-1, caspase-1 p20, GSDMD-N, and p-PERK in the THP-1 macrophage, being downregulated with the pre-treatment of GSK2656157, a PERK inhibitor. In addition, GSK2656157 inhibited the secretion of IL-1ß and IL-18, cell content release, and cell membrane rupture, as well as the decline in cell viability induced by BCG infection. Similarly, GSK2656157 treatment downregulated the expressions of pro-caspase-1, caspase-1 p20, caspase-11, IL-1ß p17, IL-18 p22, GSDMD, GSDMD-N, and p-PERK, as well as reducing fibrous tissue hyperplasia, inflammatory infiltration, and the bacterial load in the lung tissue of C57BL/6J mice infected with BCG. In conclusion, the inhibition of PERK alleviated pyroptosis induced by BCG infection, which has an effect of resisting infection.

Human Pluripotent Stem Cell-Mesenchymal Stem Cell-Derived Exosomes Promote Ovarian Granulosa Cell Proliferation and Attenuate Cell Apoptosis Induced by Cyclophosphamide in a POI-like Mouse Model.

Premature ovarian insufficiency (POI) is a complex disease which causes amenorrhea, hypergonadotropism and infertility in patients no more than 40 years old. Recently, several studies have reported that exosomes have the potential to protect ovarian function using a POI-like mouse model induced by chemotherapy drugs. In this study, the therapeutic potential of exosomes derived from human pluripotent stem cell-mesenchymal stem cells (hiMSC exosomes) was evaluated through a cyclophosphamide (CTX)-induced POI-like mouse model. POI-like pathological changes in mice were determined by serum sex-hormones levels and the available number of ovarian follicles. The expression levels of cellular proliferation proteins and apoptosis-related proteins in mouse ovarian granulosa cells were measured using immunofluorescence, immunohistochemistry and Western blotting. Notably, a positive effect on the preservation of ovarian function was evidenced, since the loss of follicles in the POI-like mouse ovaries was slowed. Additionally, hiMSC exosomes not only restored the levels of serum sex hormones, but also significantly promoted the proliferation of granulosa cells and inhibited cell apoptosis. The current study suggests that the administration of hiMSC exosomes in the ovaries can preserve female-mouse fertility.

Ror2-mediated cholesterol accumulation regulates autophagic activity within BCG-infected macrophages.

Ror2 is a primary binding partner for the non-classical Wnt signaling pathway regulator Wnt5a that plays a central role in regulating the metabolic processing of lipids within the cell. Mycobacterium tuberculosis is an intracellular pathogen that utilizes the lipid substrate cholesterol as its primary source of carbon. Cholesterol accumulation can regulate autophagy, which is in turn associated with a variety of pathological conditions. This study was designed to explore the pathways that modulate Ror2-regulated cholesterol accumulation within macrophages infected by the mycobacterium Bacillus Calmette-Guerin (BCG). BCG infection of RAW264.7 cells resulted in increased Ror2 expression, cholesterol accumulation, and autophagic activity in addition to promoting the upregulation of cholesterol synthesis-related proteins and the downregulation of cholesterol transporter proteins. Ror2 knockdown, in contrast, reversed these phenotypic changes. Treatment with T0901317 decreased the aggregation of cholesterol within cells and suppressed BCG-induced autophagy, while OX-LDL had the opposite effect. Knocking down Ror2 further reduced cholesterol levels in the context of T0901317 or OX-LDL pretreatment, alleviating BCG-induced autophagy irrespective of either of these pretreatments. Together, these data indicate that Ror2 can shape the autophagic activity induced within macrophages upon BCG infection by modulating intracellular cholesterol levels.

Wnt5a regulates autophagy in Bacille Calmette-Guérin (BCG)-Infected pulmonary epithelial cells.

Autophagy functions as a critical process that can suppress the proliferation of Mycobacterium tuberculosis (Mtb) within infected host cells. Wnt5a is a secreted protein that plays a range of physiological functions, activating several signaling pathways and thereby controlling cellular responses to particular stimuli. The importance of Wnt5a as a regulator of protection against Mtb infection, however, has yet to be fully characterized. Here, changes in murine pulmonary epithelial-like TC-1 cell morphology, autophagy, the Wnt/Ca2+ signaling pathway, and the mTOR autophagy pathway were analyzed following infection with the Mtb model pathogen Bacille Calmette-Guerin (BCG) in order to understand the regulatory role of Wnt5a in this context. These experiments revealed that Wnt5a was upregulated and autophagy was enhanced in TC-1 cells infected with BCG, and Wnt5a overexpression was found to drive BCG-induced autophagy in these cells upon infection, whereas the inhibition or knockdown of Wnt5a yielded the opposite effect. At the mechanistic level, Wnt5a was found to mediate non-canonical Wnt/Ca2+ signaling and thereby inhibit mTOR-dependent pathway activation, promoting autophagic induction within BCG-infected TC-1 cells. These data offer new insight regarding how Wnt5a influences Mtb-induced autophagy within pulmonary epithelial cells, providing a foundation for further research exploring the immunological control of this infection through the modulation of autophagy.

Prognostic Significance of Prognostic Nutritional Index in Esophageal Squamous Cell Carcinoma Patients Undergoing Radical Radiotherapy: A Propensity Score Matching Analysis.

BACKGROUND: The preoperative prognostic nutritional index (PNI) is associated with postoperative complications and long-term survival of various cancers. However, its role in esophageal squamous cell carcinoma (ESCC) is inconclusive. The aim of this study was to identify the prognostic value of PNI in predicting survival in ESCC patients undergoing radical radiotherapy. METHODS: We retrospectively reviewed 354 ESCC patients undergoing radical radiotherapy. The time-dependent receiver operating characteristics was used to determine the optimal cutoff value. The association between PNI and survival was determined by Kaplan-Meier method and Cox regression model. Propensity score matching was applied to balance the baseline characteristics. RESULTS: PNI was positively correlated with hemoglobin (P < 0.001) and prealbumin (P < 0.001). The optimal cutoff value of PNI was set at 50.5. The 5-year overall survival (OS) in low PNI group and high PNI group were 20.8% and 34.0%, respectively (P < 0.001). The 5-year progression free survival in patients with low PNI and high PNI were 15.2% and 28.5%, respectively (P = 0.001). Multivariate analysis showed that PNI was a significant predictor for OS (P = 0.038). In the PSM analysis, PNI still remained an independent predictor for OS (P = 0.015). CONCLUSIONS: The PNI is a significant and independent predictor for OS of ESCC patients undergoing radical radiotherapy.

GRAM: A GeneRAlized Model to predict the molecular effect of a non-coding variant in a cell-type specific manner.

There has been much effort to prioritize genomic variants with respect to their impact on "function". However, function is often not precisely defined: sometimes it is the disease association of a variant; on other occasions, it reflects a molecular effect on transcription or epigenetics. Here, we coupled multiple genomic predictors to build GRAM, a GeneRAlized Model, to predict a well-defined experimental target: the expression-modulating effect of a non-coding variant on its associated gene, in a transferable, cell-specific manner. Firstly, we performed feature engineering: using LASSO, a regularized linear model, we found transcription factor (TF) binding most predictive, especially for TFs that are hubs in the regulatory network; in contrast, evolutionary conservation, a popular feature in many other variant-impact predictors, has almost no contribution. Moreover, TF binding inferred from in vitro SELEX is as effective as that from in vivo ChIP-Seq. Second, we implemented GRAM integrating only SELEX features and expression profiles; thus, the program combines a universal regulatory score with an easily obtainable modifier reflecting the particular cell type. We benchmarked GRAM on large-scale MPRA datasets, achieving AUROC scores of 0.72 in GM12878 and 0.66 in a multi-cell line dataset. We then evaluated the performance of GRAM on targeted regions using luciferase assays in the MCF7 and K562 cell lines. We noted that changing the insertion position of the construct relative to the reporter gene gave very different results, highlighting the importance of carefully defining the exact prediction target of the model. Finally, we illustrated the utility of GRAM in fine-mapping causal variants and developed a practical software pipeline to carry this out. In particular, we demonstrated in specific examples how the pipeline could pinpoint variants that directly modulate gene expression within a larger linkage-disequilibrium block associated with a phenotype of interest (e.g., for an eQTL).

Effect of SIB-IMRT-based selective dose escalation of local tumor on the prognosis of patients with esophageal cancer.

BACKGROUND: To investigate the effect of SIB-IMRT-based selective dose escalation to local tumor on the prognosis of patients with esophageal cancer (EC). METHODS: A total of 302 EC patients were enrolled. The prognostic factors of the entire group were initially analyzed, and the composition ratios of the two groups and the different doses of each fraction for PTV were compared. The propensity-score matching (PSM) was carried out (1:1 ratio), and the prognostic factors for the two groups were analyzed according to the results of COX. RESULTS: The median overall survival (OS) for all patients was 30.0 months (23.495-36.505 months), and the median disease-free survival (DFS) was 21.3 months (7.698-24.902 months). In multivariate analysis, chemotherapy, cTNM stage and dose-per-fraction for the PTV were independent prognostic factors for OS (P = 0.013, 0.000, 0.028) and DFS (P = 0.033, 0.000, 0.047). Multivariate analysis of patients after PSM revealed that cTNM staging and dose-per-fraction were the independent prognostic factors for OS (P = 0.000, 0.015). Chemotherapy, cTNM staging and dose-per-fraction for the PTV were the independent prognostic factors for DFS (P = 0.025, 0.010, 0.015). There was no significant difference in grade ≥ 2 acute toxicities between the two groups. A subgroup analysis of patients with a single dose of 2 Gy and > 2 Gy in the SIB-IMRT group showed that OS and DFS of the latter were significantly better than those of the former. CONCLUSION: The selective dose escalation to local tumors based on SIB-IMRT technique can improve the survival of patients received radical radiotherapy without increasing toxicities.

Erratum to "Epithelial Cells Attenuate Toll-Like Receptor-Mediated Inflammatory Responses in Monocyte-Derived Macrophage-Like Cells to Mycobacterium tuberculosis by Modulating the PI3K/Akt/mTOR Signaling Pathway".

[This corrects the article DOI: 10.1155/2018/3685948.].

Involvement of ABC-transporters and acyltransferase 1 in intracellular cholesterol-mediated autophagy in bovine alveolar macrophages in response to the Bacillus Calmette-Guerin (BCG) infection.

BACKGROUND: Understanding pathogenic mechanisms is imperative for developing novel treatment to the tuberculosis, an important public health burden worldwide. Recent studies demonstrated that host cholesterol levels have implications in the establishment of Mycobacterium tuberculosis (M. tuberculosis, Mtb) infection in host cells, in which the intracellular cholesterol-mediated ATP-binding cassette transporters (ABC-transporters) and cholesterol acyltransferase1 (ACAT1) exhibited abilities to regulate macrophage autophagy induced by Mycobacterium bovis bacillus Calmette-Guérin (BCG). RESULTS: The results showed that a down-regulated expression of the ABC-transporters and ACAT1 in primary bovine alveolar macrophages (AMs) and murine RAW264.7 cells in response to a BCG infection. The inhibited expression of ABC-transporters and ACAT1 was associated with the reduction of intracellular free cholesterol, which in turn induced autophagy in macrophages upon to the Mycobacterial infection. These results strongly suggest an involvement of ABC-transporters and ACAT1 in intracellular cholesterol-mediated autophagy in AMs in response to BCG infection. CONCLUSION: This study thus provides an insight into into a mechanism by which the cholesterol metabolism regulated the autophagy in macrophages in response to mycobacterial infections.

Epithelial Cells Attenuate Toll-Like Receptor-Mediated Inflammatory Responses in Monocyte-Derived Macrophage-Like Cells to Mycobacterium tuberculosis by Modulating the PI3K/Akt/mTOR Signaling Pathway.

Both alveolar macrophages (AMs) and alveolar epithelial cells (AECs) are main targets of Mycobacterium tuberculosis (M. tuberculosis (Mtb)). Intercellular communications between mucosal AECs and AMs have important implications in cellular responses to exogenous insults. However, molecular mechanisms underpinning interactions responding to Mtb remain largely unknown. In this study, impacts of AECs on Toll-like receptor- (TLR-) mediated inflammatory responses of AMs to Mtb virulent strain H37Rv were interrogated using an air-liquid interface (ALI) coculture model of epithelial A549 cells and U937 monocyte-derived macrophage-like cells. Results showed that Mtb-activated TLR-mediated inflammatory responses in U937 cells were significantly alleviated when A549 cells were coinfected with H37Rv, in comparison with the infection of U937 cells alone. Mechanistically, PI3K/Akt/mTOR signaling was involved in the epithelial cell-modulated Mtb-activated TLR signaling. The epithelial cell-attenuated TLR signaling in U937s could be reversed by PI3K inhibitor LY294002 and mTOR inhibitor rapamycin, but not glycogen synthase kinase 3ß inhibitor LiCl, suggesting that the epithelially modulated-TLR signaling in macrophages was in part caused by inhibiting the TLR-triggered PI3K/Akt/mTOR signaling pathway. Together, this study demonstrates that mucosal AEC-derived signals play an important role in modulating inflammatory responses of AMs to Mtb, which thus also offers an insight into cellular communications between AECs and AMs to Mtb infections.

Are Human Translated Pseudogenes Functional?

By definition, pseudogenes are relics of former genes that no longer possess biological functions. Operationally, they are identified based on disruptions of open reading frames (ORFs) or presumed losses of promoter activities. Intriguingly, a recent human proteomic study reported peptides encoded by 107 pseudogenes. These peptides may play currently unrecognized physiological roles. Alternatively, they may have resulted from accidental translations of pseudogene transcripts and possess no function. Comparing between human and macaque orthologs, we show that the nonsynonymous to synonymous substitution rate ratio (ω) is significantly smaller for translated pseudogenes than other pseudogenes. In particular, five of 34 translated pseudogenes amenable to evolutionary analysis have ω values significantly lower than 1, indicative of the action of purifying selection. This and other findings demonstrate that some but not all translated pseudogenes have selected functions at the protein level. Hence, neither ORF disruption nor presence of protein product disproves or proves gene functionality at the protein level.

Analysis of the causes of failure after radical surgery in patients with PT3N0M0 thoracic esophageal squamous cell carcinoma and consideration of postoperative radiotherapy.

BACKGROUND: Five-year overall survival rate of TESCC after surgery is low (approximately 30% to 60%), so it is meaningful to discuss the significance of PORT. METHODS: We retrospectively collected the data of 227 patients with PT3N0M0 esophageal cancer (EC). The failure pattern after surgery was analyzed. Difference of adjuvant PORT in patients with PT3N0M0 TESCC and the appropriate population were explored based on the relevant studies. RESULTS: There were 58 cases with intrathoracic locoregional recurrence (LRR) after radical surgery and 27 cases with distant metastasis, including 10 cases of recurrence. The recurrence rate of mediastinal lymph nodes in the thoracic cavity was 50.0%. Univariate analysis revealed that compared with patients with middle and lower thoracic EC, the 3/5-year survival rate of patients with upper thoracic EC was significantly lower, accompanied with remarkably higher thoracic LRR. Compared with those with moderately- and well-differentiated TESCC, the 3/5-year survival rate of patients with poorly differentiated TESCC was significantly lower, whereas the distant metastasis rate was notably higher. Multivariate analysis revealed that different lesion locations and different pathologic differentiation were the independent prognostic factors. The lesion location and degree of differentiation were the independent influencing factors for thoracic LRR and distant metastasis, respectively. CONCLUSION: The intrathoracic LRR is the major failure pattern for patients with PT3N0M0 TESCC after conventional two-field lymphadenectomy. In addition, recurrence rate of PT3N0M0 TESCC was significantly higher in upper thoracic EC than in middle and lower thoracic EC. PORT is recommended to patients with PT3N0M0 upper TESCC.