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Summary The ever-increasing understanding of the complexity of factors and regulatory layers that contribute to immune evasion facilitates the development of immunotherapies. However, the diversity of malignant tumors limits many known mechanisms in specific genetic and epigenetic contexts, manifesting the need to discover general driver genes. Here, we have identified the m6A demethylase FTO as an essential epitranscriptomic regulator utilized by tumors to escape immune surveillance through regulation of glycolytic metabolism. We show that FTO-mediated m6A demethylation in tumor cells elevates the transcription factors c-Jun, JunB, and C/EBPβ, which allows the rewiring of glycolytic metabolism. Fto knockdown impairs the glycolytic activity of tumor cells, which restores the function of CD8+ T cells, thereby inhibiting tumor growth. Furthermore, we developed a small-molecule compound, Dac51, that can inhibit the activity of FTO, block FTO-mediated immune evasion, and synergize with checkpoint blockade for better tumor control, suggesting reprogramming RNA epitranscriptome as a potential strategy for immunotherapy.

DNA N6-methyldeoxyadenosine (6mA) is a well-known prokaryotic DNA modification that has been shown to exist and play epigenetic roles in eukaryotic DNA. Here we report that 6mA accumulates up to ∼0.1–0.2% of total deoxyadenosine during early embryogenesis of vertebrates, but diminishes to the background level with the progression of the embryo development. During this process a large fraction of 6mAs locate in repetitive regions of the genome., DNA 6mA is a poorly understood epigenetic mark present at a low abundance in eukaryotic genomes. Here the authors observe high levels in zebrafish and pig during early embryogenesis enriched to repetitive regions of the genome and followed by attenuation during development.

DNA 5hmC biomarkers to predict the outcomes to the of azacitidine prior to hi-dose cytarabine and mitoxantrone chemotherapy for patients with high-risk AML. Azacitidine (AZA), a DNA methyltransferase inhibitor and epigenetic modulator, has been combined with chemotherapy to treat high-risk AML patients for better clinical outcomes. However, the complete remission (CR) rate and prognosis remains unsatisfactory. Here, we proposed that the use of AZA for high-risk AML patients helped to sensitize leukemia cells to chemotherapy. We analyzed 45 high-risk AML patients who had received AZA treatment followed by High-dose cytarabine and mitoxantrone (AZA-HiDAC-Mito) therapy. We performed RNA-Seq and DNA 5hmC-Seq on available RNA/DNA samples. By comparing differential expressed genes between patients responded to therapy (CR or CRi, responder) and those did not (non-responder), we found that patients with better response had lower expression on ephrin receptor gene and AR pathway related genes. Using XGBoost machine learning model, we developed an 11-gene-5hmC signature to predict treatment outcomes, and it achieved 87.76% sensitivity and 87.10% specificity (AUC=0.911). The signature was also capable of distinguishing which patients would have better response to HiDAC-Mito only treatment, indicating that the signature had a potential to serve as a general predictor for intensive chemotherapy. Our findings show that DNA 5hmC patterns could be used as the prognostic biomarker for AZA-HiDAC-Mito therapy, which could guide further biomarker-based clinical trials. Citation Format: Dali HAN, Wei Zhang. Identification of DNA 5hmC decision signature biomarkers for azacitidine prior to hi-dose cytarabine and mitoxantrone chemotherapy in patients with high-risk AML [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-178.

Haimin Lin,1,2,* Dali Han,1,* Guobin Fu,3 Chengxin Liu,1 Lili Wang,4 Shumei Han,5 Bo Liu,5 Jinming Yu1 1Department of Radiation Oncology, Shandong Cancer Hospital Affiliated to Shandong University, Jinan, Shandong 250117, China; 2School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, Shandong 250200, China; 3Department of Medical Oncology, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, China; 4Department of Bone and Soft Tumor, Shandong Cancer Hospital Affiliated to Shandong University, Jinan, Shandong 250117, China; 5Department of Medical Oncology, Shandong Cancer Hospital Affiliated to Shandong University, Jinan, Shandong250117,China *These authors contributed equally to this work Purpose: The aim of this study was to assess the efficacy and safety of concurrent apatinib and docetaxel therapy vs apatinib monotherapy as third- or subsequent-line treatment for advanced gastric adenocarcinoma (GAC).Methods: Patients, who had received apatinib with or without docetaxel as third or more line therapy for advanced GAC, were retrospectively reviewed. Propensity score matching (PSM) analysis was performed to minimize the potential confounding bias. Kaplan–Meier curve and log-rank test were used to analyze the survival. Prognostic factors were estimated by Cox regression. Adverse events (AEs) were evaluated using CTCAE 4.0.Results: Thirty-four patients received concurrent therapy, whereas 31 received monotherapy. The median progression-free survival (PFS) and overall survival (OS) in monotherapy and con-therapy groups were 2.5 and 4 months (P=0.002), 3.3 and 6 months (P=0.004), respectively. After PSM, the median PFS and OS in the con-therapy group were also superior to the monotherapy group (P=0.004 and P=0.017). Cox regression suggested that Eastern Cooperative Oncology Group performance status (ECOG PS; HR =2.437, 95% CI: 1.349–4.404, P=0.003), CA199 (HR =1.001, 95% CI: 1.000–1.002, P=0.016), and treatment options (HR =0.388, 95% CI: 0.222–0.679, P=0.001) had significant effects on OS. Grade 3/4 toxicities in the monotherapy and con-therapy groups were as follows: leukopenia (0% vs 8.8%), neutropenia (3.2% vs 2.9%), anemia (9.8% vs 8.8%), thrombocytopenia (6.4% vs 2.9%), proteinuria (3.2% vs 2.9%), aminotransferase (0% vs 11.8%), hyperbilirubinemia (9.8% vs 5.9%), hypertension (9.8% vs 5.9%), hand–foot syndrome (3.2% vs 8.8%), nausea and vomiting (0% vs 11.8%), diarrhea (0% vs 5.9%), and fatigue (6.5% vs 2.9%).Conclusion: Patients with advanced GAC benefit more from concurrent apatinib and docetaxel therapy than apatinib monotherapy. Keywords: propensity score matching, progression-free survival, overall survival

Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

Non-functional parathyroid carcinoma is an exceedingly rare disease with 31 reported cases since 1909. Because of the scarce number of cases of non-functional parathyroid carcinoma, there are no evidence-based recommendations for its optimal treatment. Surgery, including en bloc resection of the carcinoma, ipsilateral thyroid lobe and isthmus together with a neck dissection only in case of lymph node involvement, is the main treatment for non-functioning parathyroid carcinoma. The patient usually has a poorer prognosis because of detection at advanced stages, the relative ineffectiveness of adjuvant treatment modalities and the lack of adequate parameters for clinical follow-up. In this report, we present a case of non-functional parathyroid carcinoma at our institution, and we review the previous literature to discuss the latest advances in the diagnosis and treatment of this rare disease.

Active DNA demethylation in mammals involves TET-mediated oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). However, genome-wide detection of 5fC at single-base resolution remains challenging. Here we present a bisulfite-free method for whole-genome analysis of 5fC, based on selective chemical labeling of 5fC and subsequent C-to-T transition during PCR. Base-resolution 5fC maps reveal limited overlap with 5hmC, with 5fC-marked regions more active than 5hmC-marked ones.

N(6)-methyladenosine (m(6)A) is the most abundant internal modification in mammalian mRNA. This modification is reversible and non-stoichiometric and adds another layer to the dynamic control of mRNA metabolism. The stability of m(6)A-modified mRNA is regulated by an m(6)A reader protein, human YTHDF2, which recognizes m(6)A and reduces the stability of target transcripts. Looking at additional functional roles for the modification, we find that another m(6)A reader protein, human YTHDF1, actively promotes protein synthesis by interacting with translation machinery. In a unified mechanism of m(6)A-based regulation in the cytoplasm, YTHDF2-mediated degradation controls the lifetime of target transcripts, whereas YTHDF1-mediated translation promotion increases translation efficiency, ensuring effective protein production from dynamic transcripts that are marked by m(6)A. Therefore, the m(6)A modification in mRNA endows gene expression with fast responses and controllable protein production through these mechanisms.

Summary N 6 -methyldeoxyadenosine (6mA or m 6 A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here, we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas . We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms.

Base-resolution maps of 5-formylcytosine and 5-carboxylcytosine reveal genome-wide DNA demethylation dynamics

5-Hydroxymethylcytosine (5hmC) is a modified form of cytosine, which has recently been found in mammalian cells and tissues. 5hmC is derived from 5-methylcytosine (5mC) by Ten-eleven translocation (TET) family protein-mediated oxidation and may regulate gene expression. Numerous affinity-based profiling methods have been developed to help understand the exact function of 5hmC in the genome. However, these methods have a relatively low resolution (~100 bp) without quantitative information of the modification percentage on each site. Here we demonstrated the detailed procedure of Tet-Assistant Bisulfite Sequencing (TAB-Seq), which can detect 5hmC at single-base resolution and quantify its abundance at each site. In this protocol, the genomic DNA is first treated with βGT and recombinant mTet1 consecutively to convert 5hmC to 5gmC and 5mC to 5caC, respectively. The treated genomic DNA can be directly applied to bisulfite treatment to detect 5hmC on specific loci or applied to whole-genome bisulfite sequencing as needed.

N(6) -methyladenosine (m(6) A) is an abundant internal modification in eukaryotic mRNA and plays regulatory roles in mRNA metabolism. However, methods to precisely locate the m(6) A modification remain limited. We present here a photo-crosslinking-assisted m(6) A sequencing strategy (PA-m(6) A-seq) to more accurately define sites with m(6) A modification. Using this strategy, we obtained a high-resolution map of m(6) A in a human transcriptome. The map resembles the general distribution pattern observed previously, and reveals new m(6) A sites at base resolution. Our results provide insight into the relationship between the methylation regions and the binding sites of RNA-binding proteins.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer featured with high intra-tumoral heterogeneity and poor prognosis. To comprehensively delineate the PDAC intra-tumoral heterogeneity and the underlying mechanism for PDAC progression, we employed single-cell RNA-seq (scRNA-seq) to acquire the transcriptomic atlas of 57,530 individual pancreatic cells from primary PDAC tumors and control pancreases, and identified diverse malignant and stromal cell types, including two ductal subtypes with abnormal and malignant gene expression profiles respectively, in PDAC. We found that the heterogenous malignant subtype was composed of several subpopulations with differential proliferative and migratory potentials. Cell trajectory analysis revealed that components of multiple tumor-related pathways and transcription factors (TFs) were differentially expressed along PDAC progression. Furthermore, we found a subset of ductal cells with unique proliferative features were associated with an inactivation state in tumor-infiltrating T cells, providing novel markers for the prediction of antitumor immune response. Together, our findings provide a valuable resource for deciphering the intra-tumoral heterogeneity in PDAC and uncover a connection between tumor intrinsic transcriptional state and T cell activation, suggesting potential biomarkers for anticancer treatment such as targeted therapy and immunotherapy.

In this Letter, a citation to ‘Fig. 1e’ has been corrected to ‘Fig. 1d’ in the sentence starting “By contrast, the anti-tumour response…”. This has been corrected online

N(6)-methyladenosine (m(6)A) is the most prevalent internal (non-cap) modification present in the messenger RNA of all higher eukaryotes. Although essential to cell viability and development, the exact role of m(6)A modification remains to be determined. The recent discovery of two m(6)A demethylases in mammalian cells highlighted the importance of m(6)A in basic biological functions and disease. Here we show that m(6)A is selectively recognized by the human YTH domain family 2 (YTHDF2) 'reader' protein to regulate mRNA degradation. We identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but which also include non-coding RNAs, with a conserved core motif of G(m(6)A)C. We further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies. The carboxy-terminal domain of YTHDF2 selectively binds to m(6)A-containing mRNA, whereas the amino-terminal domain is responsible for the localization of the YTHDF2-mRNA complex to cellular RNA decay sites. Our results indicate that the dynamic m(6)A modification is recognized by selectively binding proteins to affect the translation status and lifetime of mRNA.

Long non-coding RNAs (lncRNAs) have been shown to participate in cancer progression. In the present study, we explored the potential roles of lncRNA-PVT1 in the development process of colorectal cancer (CRC) via miR-455. We found that PVT1 is up-regulated in human CRC tissues compared to adjacent normal tissues. A functional study showed that the silencing of PVT1 expression by siRNAs inhibited cell proliferation, migration and invasion, whereas the overexpression of PVT1 accelerated cell proliferation, migration and invasion in vitro. A mechanistic study indicated PVT1 regulated the growth of CRC tumors by acting as a competing endogenous RNAs (ceRNA) and negatively regulated miR-455. Furthermore, we discovered that RUNX2, a functional transcription factor in CRC, up-regulated PVT1 expression. Therefore, our study suggested that the RUNX2/PVT1/miR-455 regulatory axis plays an important role in CRC tumorigenesis and may be a therapeutic target for the treatment of CRC.

The ribose of RNA nucleotides can be 2'-O-methylated (Nm). Despite advances in high-throughput detection, the inert chemical nature of Nm still limits sensitivity and precludes mapping in mRNA. We leveraged the differential reactivity of 2'-O-methylated and 2'-hydroxylated nucleosides to periodate oxidation to develop Nm-seq, a sensitive method for transcriptome-wide mapping of Nm with base precision. Nm-seq uncovered thousands of Nm sites in human mRNA with features suggesting functional roles.

We present a highly sensitive and selective chemical labeling and capture approach for genome-wide profiling of 5-hydroxylmethylcytosine (5hmC) using DNA isolated from ~1,000 cells (nano-hmC-Seal). Using this technology, we assessed 5hmC occupancy and dynamics across different stages of hematopoietic differentiation. Nano-hmC-Seal profiling of purified Tet2-mutant acute myeloid leukemia (AML) murine stem cells allowed us to identify leukemia-specific, differentially hydroxymethylated regions that harbor known and novel candidate disease-specific target genes with differential 5hmC peaks compared to normal stem cells. The change of 5hmC patterns in AML strongly correlates with differential gene expression, demonstrating the importance of dynamic alterations of 5hmC in regulating transcription in AML. Together, covalent 5hmC labeling offers an effective approach to study and detect DNA methylation dynamics in in vivo disease models and in limited clinical samples.

Transfer RNA (tRNA) is a central component of protein synthesis and cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and their decreased usage in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new regulatory mechanism of post-transcriptional gene expression.

Nat. Methods 14, 695–698 (2017); published online 15 May 2017; corrected after print 28 February 2018 We were alerted by readers that the reported Nm consensus sequence in mRNA matches the 3′-adaptor sequence used in sequencing library preparations, and this could be caused by mispriming1. In our approach, the majority of RNA fragments without Nm at the 3′ end are blocked from ligating to the 3′ adaptor because of the presence of 3′ phosphate from the last oxidation elimination step (OE) (Fig. 1a of the original paper), while Nm sites accumulate at the 3′ ends (Supplementary Figs. 1 and 2; Supplementary Notes 1 and 2). However, because of the low Nm abundance in messenger RNA (mRNA), only very limited amounts of mRNA fragments carry 3′ Nm and thus can be successfully ligated to the 3′ adaptor. Mispriming could occur if the 3′ end of the reverse transcription (RT) primer hybridizes to a few bases of the 5′-ligated RNA (Fig. 1a). Although our method effectively identifies Nm sites in abundant ribosomal RNA (rRNA, Supplementary Fig. 1), its application to less abundant mRNA can be contaminated by mispriming, leading to false-positive Nm sites and the erroneous AGAUC motif on mRNA (original Fig. 2d), which corresponds to the 5′-end sequence of the 3′ adaptor.

Summary Epigenetic modifications are critical determinants of cellular and developmental states. Epigenetic changes, such as decreased H3K27me3 histone methylation on insulin/IGF1 genes, have been previously shown to modulate lifespan through gene expression regulation. However, global epigenetic changes during aging and their biological functions, if any, remain elusive. Here, we examined the histone modification H3K4 dimethylation (H3K4me2) in the prefrontal cortex of individual rhesus macaques at different ages by chromatin immunoprecipitation, followed by deep sequencing (ChIP-seq) at the whole genome level. Through integrative analysis of the ChIP-seq profiles with gene expression data, we found that H3K4me2 increased at promoters and enhancers globally during postnatal development and aging, and those that correspond to gene expression changes in cis are enriched for stress responses, such as the DNA damage response. This suggests that metabolic and environmental stresses experienced by an organism are associated with the progressive opening of chromatin. In support of this, we also observed increased expression of two H3K4 methyltransferases, SETD7 and DPY30, in aged macaque brain.

Rhesus macaque is a widely used primate model organism. Its genome annotations are however still largely comparative computational predictions derived mainly from human genes, which precludes studies on the macaque-specific genes, gene isoforms or their regulations. Here we took advantage of histone H3 lysine 4 trimethylation (H3K4me3)’s ability to mark transcription start sites (TSSs) and the recently developed ChIP-Seq and RNA-Seq technology to survey the transcript structures. We generated 14 013 757 sequence tags by H3K4me3 ChIP-Seq and obtained 17 322 358 paired end reads for mRNA, and 10 698 419 short reads for sRNA from the macaque brain. By integrating these data with genomic sequence features and extending and improving a state-of-the-art TSS prediction algorithm, we ab initio predicted and verified 17 933 of previously electronically annotated TSSs at 500-bp resolution. We also predicted approximately 10 000 novel TSSs. These provide an important rich resource for close examination of the species-specific transcript structures and transcription regulations in the Rhesus macaque genome. Our approach exemplifies a relatively inexpensive way to generate a reasonably reliable TSS map for a large genome. It may serve as a guiding example for similar genome annotation efforts targeted at other model organisms.

The primary aim of this study was to use serial 18F-3′-deoxy-3′-fluorothymidine (FLT) PET/CT to measure tumor cell proliferation during radiotherapy of squamous cell carcinoma (SCC) of the esophagus. Methods: Twenty-one patients with inoperable locally advanced SCC of the esophagus underwent serial 18F-FLT PET/CT during radiotherapy. Each patient received a pretreatment scan, followed by 1–3 scans after delivery of 2, 6, 10, 20, 30, 40, 50, or 60 Gy to the tumor. Results: Among the 19 patients who completed radiotherapy without interruption, parameters reflecting 18F-FLT uptake in the tumor (i.e., maximum tumor standardized uptake value [SUVmax] and proliferation target volume) decreased steadily. All patients demonstrated an almost complete absence of proliferating esophageal tumor after 30 Gy and a complete absence after 40 Gy. In the 2 patients whose radiotherapy course was interrupted, 18F-FLT uptake in the tumor was greater after the interruption than before the interruption. Marked early reduction of 18F-FLT uptake in irradiated bone marrow was observed in all patients, even after only 2 Gy. All showed a complete absence of proliferating marrow in irradiated regions after 10 Gy. Both patients who underwent scans after completing the entire radiotherapy course showed no tumor uptake on 18F-FLT PET/CT but high uptake on 18F-FDG PET/CT. Pathologic examination of these regions revealed inflammatory infiltrates but no residual tumor. Conclusion:18F-FLT uptake can be used to monitor the biologic response of esophageal SCC and normal tissue to radiotherapy. Increased uptake of 18F-FLT after treatment interruptions may reflect accelerated repopulation. 18F-FLT PET/CT may have an advantage over 18F-FDG PET/CT in differentiating inflammation from tumor.

In the active cytosine demethylation pathway, 5-methylcytosine (5mC) is oxidized sequentially to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Thymine DNA glycosylase (TDG) selectively excises 5fC and 5caC but not cytosine (C), 5mC, and 5hmC. We propose that the electron-withdrawing properties of -CHO and -COOH in 5fC and 5caC increase N3 acidity, leading to weakened hydrogen bonding and reduced base pair stability relative to C, 5mC, and 5hmC, thereby facilitating the selective recognition of 5fC and 5caC by TDG. Through (13)C NMR, we measured the pKa at N3 of 5fC as 2.4 and the two pKa's of 5caC as 2.1 and 4.2. We used isotope-edited IR spectroscopy coupled with density functional theory (DFT) calculations to site-specifically assign the more acidic pKa of 5caC to protonation at N3, indicating that N3 acidity is increased in 5fC and 5caC relative to C. IR and UV melting studies of self-complementary DNA oligomers confirm reduced stability for 5fC-G and 5caC-G base pairs. Furthermore, while the 5fC-G base pair stability is insensitive to pH, the 5caC-G stability is reduced as pH decreases and the carboxyl group is increasingly protonated. Despite suggestions that 5fC and 5caC may exist in rare tautomeric structures which form wobble GC base pairs, our two-dimensional infrared (2D IR) spectroscopy of 5fC and 5caC free nucleosides confirms that both bases are predominantly in the canonical amino-keto form. Taken together, these findings support our model that weakened base pairing ability for 5fC and 5caC in dsDNA contributes to their selective recognition by TDG.

Bile acid plays an important role in regulating blood glucose, lipid and energy metabolism. The present study was implemented to determine the effect of duodenal-jejunal bypass (DJB) on FXR, TGR-5expression in terminal ileum and its bile acid-related mechanism on glucose and lipid metabolism. Immunohistochemistry was used to detect relative gene or protein expression in liver and intestine. Firstly, we found that expression of FXR in liver and terminal ileum of DJB group was significantly higher than that in S-DJB group (P

Accurate delineation of clinical target volume (CTV) is critical in the effective management of squamous cell carcinoma (SCC) of esophagus using radiation therapy. Accurate delineation may improve the probability of local control and reduce the risk of complications. However, there are no consistent standards on the proper size of the margins added to the gross tumor volume (GTV). Different institutions and radiation oncologists have discordant opinions. In this paper, we review pathological and clinical outcomes to determine the most appropriate CTV for squamous cell carcinomas (SCC) of esophagus. The CTV for esophageal carcinoma should ensure that all subclinical lesions are encompassed regardless of the physical distance. The most precise method for delineating a reasonable CTV is to combine advanced imaging techniques, such as PET/CT and EUS, which allows the detection and prediction of subclinical lesions based on tumor characteristics such as the pathological type, differentiation, T disease, length and lymph node status.

Gene expression can be regulated post-transcriptionally through dynamic and reversible RNA modifications. A recent noteworthy example is N(6)-methyladenosine (m(6)A), which affects messenger RNA (mRNA) localization, stability, translation and splicing. Here we report on a new mRNA modification, N(1)-methyladenosine (m(1)A), that occurs on thousands of different gene transcripts in eukaryotic cells, from yeast to mammals, at an estimated average transcript stoichiometry of 20% in humans. Employing newly developed sequencing approaches, we show that m(1)A is enriched around the start codon upstream of the first splice site: it preferentially decorates more structured regions around canonical and alternative translation initiation sites, is dynamic in response to physiological conditions, and correlates positively with protein production. These unique features are highly conserved in mouse and human cells, strongly indicating a functional role for m(1)A in promoting translation of methylated mRNA.

N(6)-methyladenosine (m(6)A) is the most prevalent and reversible internal modification in mammalian messenger and noncoding RNAs. We report here that human methyltransferase-like 14 (METTL14) catalyzes m(6)A RNA methylation. Together with METTL3, the only previously known m(6)A methyltransferase, these two proteins form a stable heterodimer core complex of METTL3-METTL14 that functions in cellular m(6)A deposition on mammalian nuclear RNAs. WTAP, a mammalian splicing factor, can interact with this complex and affect this methylation.

Background To validate a gradient-based segmentation method for gross tumor volume(GTV) delineation on 8F-fluorothymidine (FLT)positron emission tomography (PET)/ computer tomography (CT) in esophageal squamous cell cancer through pathologic specimen, in comparison with standardized uptake values (SUV) threshold-based methods and CT. The corresponding impact of this GTV delineation method on treatment planning was evaluated. Methods and materials Ten patients with esophageal squamous cell cancer were enrolled. Before radical surgery, all patients underwent FLT-PET/CT. GTVs were delineated by using four methods. GTVGRAD, GTV1.4 and GTV30%max were segmented on FLT PET using a gradient-based method, a fixed threshold of 1.4 SUV and 30% of SUVmax, respectively. GTVCT was based on CT data alone. The maximum longitudinal tumor length of each segmented GTV was compared with the measured tumor length of the pathologic gross tumor length (LPath). GTVGRAD, GTV1.4 and GTV30%max were compared with GTVCT by overlap index. Two radiotherapy plannings (planGRAD) and (planCT) were designed for each patient based on GTVGRAD and GTVCT. The dose-volume parameters for target volume and normal tissues, CI and HI of planGRAD and planCT were compared. Results The mean ± standard deviation of LPath was 6.47 ± 2.70 cm. The mean ± standard deviation of LGRAD,L1.4, L30%max and LCT were 6.22 ± 2.61, 6.23 ± 2.80, 5.95 ± 2.50,7.17 ± 2.28 cm, respectively. The Pearson correlation coefficients between LPath and each segmentation method were 0.989, 0.920, 0.920 and 0.862, respectively. The overlap indices of GTVGRAD, GTV1.4, GTV30%max when compared with GTVCT were 0.75 ± 0.12, 0.71 ± 0.12, 0.57 ± 0.10, respectively. The V5, V10, V20, V30 and mean dose of total-lung,V30 and mean dose of heart of planGRAD were significantly lower than planCT. Conclusions The gradient-based method provided the closest estimation of target length. The radiotherapy plannings based on the gradient-based segmentation method reduced the irradiated volume of lung, heart in comparison to CT.

Summary Inhibition of cytosolic DNA sensing represents a strategy that tumor cells use for immune evasion, but the underlying mechanisms are unclear. Here we have shown that CD47-signal regulatory protein α (SIRPα) axis dictates the fate of ingested DNA in DCs for immune evasion. Although macrophages were more potent in uptaking tumor DNA, increase of DNA sensing by blocking the interaction of SIRPα with CD47 preferentially occurred in dendritic cells (DCs) but not in macrophages. Mechanistically, CD47 blockade enabled the activation of NADPH oxidase NOX2 in DCs, which in turn inhibited phagosomal acidification and reduced the degradation of tumor mitochondrial DNA (mtDNA) in DCs. mtDNA was recognized by cyclic-GMP-AMP synthase (cGAS) in the DC cytosol, contributing to type I interferon (IFN) production and antitumor adaptive immunity. Thus, our findings have demonstrated how tumor cells inhibit innate sensing in DCs and suggested that the CD47-SIRPα axis is critical for DC-driven antitumor immunity.

The primary aim of this prospective study was to investigate the value of (99m)Tc-methoxyisobutylisonitrile (MIBI) single photon emission computed tomography (SPECT) gated myocardial perfusion imaging (GMPI) in the detection of radiation-induced heart disease (RIHD) as early as during radiotherapy (RT) for oesophageal cancer (EC). The second aim was to analyse the correlation between cardiac toxicity and the dose-volume factors.The (99m)Tc-MIBI SPECT GMPI was performed both pre-RT and during RT (40Gray). The results of the SPECT were quantitatively analysed with QGS/QPS software and read by two experienced nuclear medicine physicians. The correlation between the changes in the SPECT parameters and the RT dosimetric data was analysed.Eighteen patients with locally advanced EC were enrolled in the study. Compared with the baseline, the imaging during RT showed not only significant decreases in the wall motion (WM) (1/20 segments), wall thickening (WT) (2/20 segments), end-diastolic perfusion (EDP) (5/20 segments) and end-systolic perfusion (ESP) (8/20 segments) (p0.05) but also a significant increase in the heart rate (74.63±7.79 vs 81.49±9.90, p=0.036). New myocardial perfusion defects were observed in 8 of the 18 patients. The V37-V40 was significantly higher (p0.05) in the patients with the new perfusion defects during RT than in the patients who did not exhibit these defects.Radiotherapy for EC induces cardiac damage from an early stage. (99m)Tc-MIBI SPECT GMPI can detect the occurrence of cardiac impairment during RT. The WM, WT, EDP and ESP may be valuable as early indicators of RIHD. The percentage of the heart volume that receives a high dose is an important factor that is correlated with RIHD.

SummaryEpigenetic modifications are thought to be important for gene expression changes during development and aging. However, besides the Sir2 histone deacetylase in somatic tissues and H3K4 trimethylation in germlines, there is scant evidence implicating epigenetic regulations in aging. The insulin/IGF-1 signaling (IIS) pathway is a major life span regulatory pathway. Here, we show that progressive increases in gene expression and loss of H3K27me3 on IIS components are due, at least in part, to increased activity of the H3K27 demethylase UTX-1 during aging. RNAi of the utx-1 gene extended the mean life span of C. elegans by ∼30%, dependent on DAF-16 activity and not additive in daf-2 mutants. The loss of utx-1 increased H3K27me3 on the Igf1r/daf-2 gene and decreased IIS activity, leading to a more “naive” epigenetic state. Like stem cell reprogramming, our results suggest that reestablishment of epigenetic marks lost during aging might help “reset” the developmental age of animal cells.