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Background: Upper gastrointestinal cancer (UGC) is an important cause of cancer death in China, with low five-year survival rates due to the majority of UGC patients being diagnosed at an advanced stage. Therefore, there is an urgent need to develop cost-effective, reliable and non-invasive methods for the early detection of UGC. Methods: A novel plasma-based methylation panel combining simultaneous detection of three methylated biomarkers (ELMO1, ZNF582 and TFPI2) and an internal control gene were developed and used to examine plasma samples from 186 UGC patients and 190 control subjects. Results: The results indicated excellent PCR amplification efficiency and reproducibility of ELMO1, ZNF582 and TFPI2 in the range of 10–100,000 copies per PCR reaction of fully methylated genomic DNA. The methylation levels of ELMO1, ZNF582 and TFPI2 were significantly higher in UGC samples than those in control subjects. The sensitivities of ELMO1, ZNF582 and TFPI2 alone for UGC detection were 32.3%, 61.3% and 30.6%, respectively; when three markers were combined, the sensitivity was improved to 71.0%, with a specificity of 90.0%, and the area under the curve (AUC) was 0.870 (95% CI: 0.832–0.902). Conclusion: Methylated ELMO1, ZNF582 and TFPI2 were specific for UGC and the three-methylated gene panel provided an alternative non-invasive choice for UGC early detection.

Following fertilization, the zygotic genome is activated through a process termed zygotic genome activation (ZGA), which enables zygotic gene products to replace the maternal products and initiates early embryonic development. During the ZGA period, the embryonic epigenome experiences extensive recodifications. The H3K27me3 demethylase UTX is essential for post-implantation embryonic development. However, it remains unclear whether UTX participates in preimplantation development, especially during the ZGA process. In the present study, we showed that either knockdown or overexpression of UTX led to embryonic development retardation, whereas simultaneous depletion of UTX and overexpression of ZSCAN4D rescued the embryonic development, indicating that UTX positively regulated Zscan4d expression. Using a transgenic mice model, we also found that UTX was required for preimplantation embryonic development. In conclusion, these results indicate that UTX functions as a novel regulator and plays critical roles during ZGA in addition to early embryonic development.

e16096 Background: Gastrointestinal cancers (colorectal cancer, gastric cancer and esophageal cancer) are the most prevalent malignant tumors in China, resulting in 975,537 new cases and 958,675 deaths in 2020. Currently, the gold standard for the early detection of these cancers remains to be gastrointestinal endoscopy. However, the invasiveness, low throughput and high cost of these procedures have prevented its wide acceptance by Chinese population. The objective of this study was to develop a liquid biopsy test based on the detection of multiple DNA methylation biomarkers (SpecGastro test) and evaluate its clinical performance for early detection of gastrointestinal cancers. Methods: Two hundred and eighty-two gastrointestinal cancer patients (98 colorectal cancers, 136 gastric cancers and 48 esophageal cancers) and 195 control subjects were included. Ten milliliters of peripheral blood was drawn and 3.5 mL plasma was separated for cell-free DNA extraction and bisulfite conversion. The converted DNA was subsequently examined by SpecGastro test, a multiplex qPCR assay that detected six methylation biomarkers and one internal control. Results: Overall sensitivity for SpecGastro test was 76.6% (95% CI: 71.1-81.3%) with a specificity of 89.2% (95% CI: 83.8-93.1%). Sensitivities for colorectal cancer, gastric cancer and esophageal cancer were 87.8% (95% CI: 79.2-93.2%), 69.9% (95% CI: 61.3-77.3%) and 72.9% (95% CI: 57.9-84.3%), respectively. A high AUC of 0.903 (95% CI: 0.873-0.928) indicated that SpecGastro test distinguished gastrointestinal cancer patients from control subjects with high accuracy. Conclusions: Our analysis demonstrated the utility of SpecGastro test to detect three types of gastrointestinal cancers with high sensitivity and specificity. These results supported the feasibility of this blood-based SpecGastro test as a complementary tool to the existing single-cancer early detection tests.

Cloned animals generated by somatic cell nuclear transfer (SCNT) have been reported for many years; however, SCNT is extremely inefficient, and zygotic genome activation (ZGA) is required for SCNT-mediated somatic cell reprogramming. To identify candidate factors that facilitate ZGA in SCNT-mediated reprogramming, we performed siRNA-repressor and mRNA-inducer screenings, which reveal Dux, Dppa2, and Dppa4 as key factors enhancing ZGA in SCNT. We show that direct injection of ZGA inducers has no significant effect on SCNT blastocyst formation; however, following the establishment of an inducible Dux transgenic mouse model, we demonstrate that transient overexpression of Dux not only improves SCNT efficiency but also increases that of chemically induced pluripotent stem cell reprogramming. Moreover, transcriptome profiling reveals that Dux-treated SCNT embryos are similar to fertilized embryos. Furthermore, transient overexpression of Dux combined with inactivation of DNA methyltransferases (Dnmts) further promotes the full embryonic development of SCNT-derived animals. These findings enhance our understanding of ZGA-regulator function in somatic reprogramming.

N6-methyladenosine (m6A) methylation is the most common and abundant modification on mammalian messenger RNA (mRNA) and regulates the pluripotency of embryonic stem cells (ESCs). Research has shown that melatonin plays a fundamental role in DNA and histone modifications. However, the effect of melatonin on RNA modification is unknown. Here, for the first time, we investigated the effect of melatonin on m6A modifications in long-term-cultured ESCs. Pluripotency studies indicated that 10 μmol/L melatonin sufficiently maintained ESCs with stemness features over 45 passages (more than 90 days). Notably, treatment of ESCs with melatonin led to a significant decrease in the nuclear presence of m6A methyltransferase complex and decreased global m6A modification. Depletion of melatonin receptor 1 (MT1) by CRISPR/Cas9 significantly reduced the effects of melatonin on ESC pluripotency and m6A modification. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) revealed that melatonin promotes stabilization of core pluripotency factors, such as Nanog, Sox2, Klf4, and c-Myc, by preventing m6A-dependent mRNA decay. Using cell signaling pathway profiling systems, melatonin was shown to regulate m6A modification predominantly through the MT1-JAK2/STAT3-Zfp217 signal axis. This study reveals a new dimension regarding melatonin regulation of gene expression at the RNA level.

Oryza longistaminata, a perennial wild species, is widely distributed in the African continent. It has strong tolerance to biotic and abiotic stresses, and high biomass production on poor soils. Chlorophyll biosynthesis is important for photosynthesis in rice. However, the chlorophyll biosynthesis and related gene profiles of O. longistaminata and its descendants remained unclear. Here, the F1 generation of O. sativa and O. longistaminata were obtained. Then, the comparative analysis morphology, anatomical structure, and transcriptional regulatory networks of chlorophyll biosynthesis were detected and analyzed. Results showed that the F1 generation has obvious long awn, similar with that of the male parent. The purple color of the long awn is different from that of the male parent. Microstructural results showed that the flag leaves of F1 have large mesophyll cell gaps in the upper- and lower-positions, small mesophyll cell gaps in the middle position, and more chloroplasts. Increased chlorophyll content was also observed in the F1 generation. In the lower-position flag leaves, the total chlorophyll contents of F1 were 1.55 and 1.5 times those of O. sativa and O. longistaminata, respectively. POR, MgCH and HEMA1 showed higher expression levels than the other related genes selected in the chlorophyll biosynthesis pathway. The HEMA1 expression level in the middle-position flag leaves of O. longistaminata was the highest, and it was 2.83 and 2.51 times that of O. sativa and F1, respectively. The expression level of DVR gene in lower-position flag leaves of F1 were 93.16% and 95.06% lower than those of O. sativa and O. longistaminata, respectively. This study provided a potential reference for studying the photosynthesis and heterosis utilization of O. longistaminata.

The efficiency of somatic cell nuclear transfer (SCNT) reprogramming is extremely low in terms of production of cloned animals. Here, we found that telomere rejuvenation is a critical event for SCNT reprogramming. Through small-molecule screening, we identified that melatonin significantly improved the

Cloned animal have been reported by somatic cell nuclear transfer (SCNT) for many years. However, the SCNT is extremely inefficient, and zygotic genome activation (ZGA) is required for SCNT and chemical mediated reprogramming. To identify candidate factors that facilitate ZGA in reprogramming, we performed siRNA-repressor and mRNA-inducer screening, which revealed Dux, Dppa2, and Dppa4 as key factors enhancing the ZGA in SCNT. Direct injection of ZGA-inducers had no significant effect on the SCNT blastocyst formation, and even destroyed the ZGA. Through a inducible Dux transgenic mouse model, we demonstrate that transient overexpression of Dux not only improved SCNT efficiency, but also increased the efficiency of chemical reprogramming. Transcriptome profiling revealed that Dux treated SCNT embryos were similar to fertilized embryos. Furthermore, transient overexpression of Dux combined with inactivation of DNA methyltransferases (Dnmts) further promote the overall development of SCNT-derived animals. These findings enhance our understanding of ZGA-regulators in somatic reprogramming.

Linoleic acid (18 : 2, n-6) and α-linolenic acid (18 : 3, n-3) are polyunsaturated fatty acids (PUFAs), which are essential for mammalian health, development and growth. However, the majority of mammals, including humans, are incapable of synthesizing n-6 and n-3 PUFAs. Mammals must obtain n-6 and n-3 PUFAs from their diet. Fatty acid desaturase (Fad) plays a critical role in plant PUFA biosynthesis. Therefore, we generated plant-derived Fad3 single and Fad2–Fad3 double transgenic mice. Compared with wild-type mice, we found that PUFA levels were greatly increased in the single and double transgenic mice by measuring PUFA levels. Moreover, the concentration of n-6 and n-3 PUFAs in the Fad2–Fad3 double transgenic mice were greater than that in the Fad3 single transgenic mice. These results demonstrated that the plant-derived Fad2 and Fad3 genes can be expressed in mammals. To clarify the mechanism for Fad2 and Fad3 genes in transgenic mice, we measured the PUFAs synthesis-related genes. Compared with wild-type mice, these Fad transgenic mice have their own n-3 and n-6 PUFAs biosynthetic pathways. Thus, we have established a simple and efficient method for in vivo synthesis of PUFAs.

Despite the success of the production of animals by somatic cell nuclear transfer (SCNT) in many species, the method is limited by a low efficiency. After zygotic genome activation (ZGA), a large number of endogenous retroviruses (ERVs) are expressed, including the murine endogenous retrovirus-L (MuERVL/MERVL). In this study, we generated a series of MERVL-reporter mouse strains to detect the ZGA event in embryos. We found that the majority of SCNT embryos exhibited ZGA failure, and histone H3 lysine 27 trimethylation (H3K27me3) prevented SCNT reprogramming. Overexpression of the H3K27me3-specific demethylase KDM6A, but not KDM6B, improved the efficiency of SCNT. Conversely, knockdown KDM6B not only facilitate ZGA, but also impede ectopic Xist expression in SCNT reprogramming. Furthermore, the knockdown of KDM6B increased the rate of SCNT-derived Duchenne muscular dystrophy embryonic stem cell establishment, indicate that these results not only provide insight into the mechanisms underlying failures of SCNT, but also may extend the applications of SCNT.

Despite the success of animal cloning by somatic cell nuclear transfer (SCNT) in many species, the method is limited by its low efficiency. After zygotic genome activation (ZGA) during mouse development, a large number of endogenous retroviruses (ERVs) are expressed, including the murine endogenous retrovirus‐L (MuERVL/MERVL). In this study, we generate a series of MERVL reporter mouse strains to detect the ZGA event in embryos. We show that the majority of SCNT embryos do not undergo ZGA, and H3K27me3 prevents SCNT reprogramming. Overexpression of the H3K27me3‐specific demethylase KDM6A, but not of KDM6B, improves the efficiency of SCNT. Conversely, knockdown of KDM6B not only facilitates ZGA, but also impedes ectopic Xist expression in SCNT reprogramming. Furthermore, knockdown of KDM6B increases the rate of SCNT‐derived embryonic stem cells from Duchenne muscular dystrophy embryos. These results not only provide insight into the mechanisms underlying failures of SCNT, but also may extend the applications of SCNT.

Linoleic acid (18 : 2, n-6) and α-linolenic acid (18 : 3, n-3) are polyunsaturated fatty acids (PUFAs), which are essential for mammalian health, development and growth. However, the majority of mammals, including humans, are incapable of synthesizing n-6 and n-3 PUFAs. Mammals must obtain n-6 and n-3 PUFAs from their diet. Fatty acid desaturase (Fad) plays a critical role in plant PUFA biosynthesis. Therefore, we generated plant-derived Fad3 single and Fad2–Fad3 double transgenic mice. Compared with wild-type mice, we found that PUFA levels were greatly increased in the single and double transgenic mice by measuring PUFA levels. Moreover, the concentration of n-6 and n-3 PUFAs in the Fad2–Fad3 double transgenic mice were greater than in the Fad3 single transgenic mice. These results demonstrate that the plant-derived Fad2 and Fad3 genes can be expressed in mammals. To clarify the mechanism for Fad2 and Fad3 genes in transgenic mice, we measured the PUFAs synthesis-related genes. Compared with wild-type mice, these Fad transgenic mice have their own n-3 and n-6 PUFAs biosynthetic pathways. Thus, we have established a simple and efficient method for in vivo synthesis of PUFAs.

During the process of embryonic development in mammals, epigenetic modifications must be erased and reconstructed. In particular, the trimethylation of histone 3 lysine 27 (H3K27me3) is associated with gene-specific transcriptional repression and contributes to the maintenance of the pluripotent embryos. In this study, we determined that the global levels of the H3K27me3 marker were elevated in MII oocyte chromatin and decrease to minimal levels at the 8-cell and morula stages. When the blastocyst hatched, H3K27me3 was re-established in the inner cell mass. We also determined that H3K27me3-specific demethylases, UTX and JMJD3, were observed at high transcript and protein levels in mouse preimplantation embryos. In the activated oocytes, when the H3K27me3 disappeared at the 8-cell stage, the UTX (but not JMJD3) protein levels were undetectable. Using RNA interference, we suppressed UTX and JMJD3 gene expression in the embryos and determined that the functions of UTX and JMJD3 were complementary. When JMJD3 levels were decreased by RNA interference, the embryo development rate and quality were improved, but the knockdown of UTX produced the opposite results. Understanding the epigenetic mechanisms controlling preimplantation development is critical to comprehending the basis of embryonic development and to devise methods and approaches to treat infertility.

The development process of synchronous pulley under the circle of UG is introduced in this paper. Arc tooth synchronous pulley is taken for an example. The automatic modeling module of arc tooth synchronous pulley is eveloped by UG/Open GRIP. In this module, users can get the synchronous pulley solid model they need just by entering the corresponding parameters on the man-machine interface.