Recently, RNA-guided genome editing using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) system has been applied to edit the plant genome in ...several herbaceous plant species. However, it remains unknown whether this system can be used for genome editing in woody plants. In this study, we describe the genome editing and targeted gene mutation in a woody species, Populus tomentosa Carr. via the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed to target with distinct poplar genomic sites of the phytoene desaturase gene 8 (PtoPDS) which are followed by the protospacer-adjacent motif (PAM). After Agrobacterium-mediated transformation, obvious albino phenotype was observed in transgenic poplar plants. By analyzing the RNA-guided genome-editing events, 30 out of 59 PCR clones were homozygous mutants, 2 out of 59 were heterozygous mutants and the mutation efficiency at these target sites was estimated to be 51.7%. Our data demonstrate that the Cas9/sgRNA system can be exploited to precisely edit genomic sequence and effectively create knockout mutations in woody plants.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Summary
The secondary cell wall is an important carbon sink in higher plants and its biosynthesis requires coordination of metabolic fluxes in the phenylpropanoid pathway. In Arabidopsis (Arabidopsis ...thaliana), MYB75 and the KNOX transcription factor KNAT7 form functional complexes to regulate secondary cell wall formation in the inflorescence stem. However, the molecular mechanism by which these transcription factors control different branches of the phenylpropanoid pathway remains poorly understood in woody species. We isolated an R2R3‐MYB transcription factor MYB6 from Populus tomentosa and determined that it was expressed predominately in young leaves. Overexpression of MYB6 in transgenic poplar upregulated flavonoid biosynthetic gene expression, resulting in significantly increased accumulation of anthocyanin and proanthocyanidins. MYB6‐overexpression plants showed reduced secondary cell wall deposition, accompanied by repressed expression of secondary cell wall biosynthetic genes. We further showed that MYB6 interacted physically with KNAT7 and formed functional complexes that acted to repress secondary cell wall development in poplar and Arabidopsis. The results provide an insight into the transcriptional mechanisms involved in the regulation of the metabolic fluxes between the flavonoid and lignin biosynthetic pathways in poplar.
Significance Statement
The poplar MYB6 transcription factor is involved in the positive regulation of anthocyanin and proanthocyanidin biosynthesis but the inhibition of secondary cell wall formation with KNAT7 repressor.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Key message
A novel R2R3 MYB transcription factor
PtrMYB57
interacted with bHLH131 and PtrTTG1 to form the MBW complex and negatively regulated the biosynthesis of both anthocyanins and PAs in ...poplar.
R2R3-MYB transcription factors (TFs) are important regulators of secondary metabolite biosynthesis in woody species. A series of R2R3-MYB TFs involved in anthocyanin and proanthocyanidin (PA) biosynthesis have been identified in poplar. In this study, we report the identification and characterization of a subgroup 4 MYB member PtrMYB57, which contains a repressor domain (LxLxL) at the C-terminal end.
PtrMYB57
encodes an R2R3 MYB protein localized in the nucleus and is predominantly expressed in mature leaves. Transgenic poplar overexpressing
PtrMYB57
showed a reduction in anthocyanin and PA accumulation compared to wild-type plants. By contrast, a high anthocyanin and PA phenotype was observed in
Ptrmyb57
mutants generated by the CRISPR/Cas9 system. Furthermore, transient expression assays revealed that PtrMYB57 interacted with bHLH131 (bHLH) and PtrTTG1 (WDR) to form the MBW complex and bound to the flavonoid gene promoters, leading to inhibition of these promoters. Taken together, our results suggest that PtrMYB57 plays a negative role in the regulation of anthocyanin and PA biosynthesis in poplar.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Circulating tumor DNA (ctDNA) has emerged as a useful diagnostic and prognostic biomarker in many cancers. Here, we conducted a study to investigate the potential use of ctDNA methylation markers for ...the diagnosis and prognostication of colorectal cancer (CRC) and used a prospective cohort to validate their effectiveness in screening patients at high risk of CRC. We first identified CRC-specific methylation signatures by comparing CRC tissues to normal blood leukocytes. Then, we applied a machine learning algorithm to develop a predictive diagnostic and a prognostic model using cell-free DNA (cfDNA) samples from a cohort of 801 patients with CRC and 1021 normal controls. The obtained diagnostic prediction model discriminated patients with CRC from normal controls with high accuracy (area under curve = 0.96). The prognostic prediction model also effectively predicted the prognosis and survival of patients with CRC (
< 0.001). In addition, we generated a ctDNA-based molecular classification of CRC using an unsupervised clustering method and obtained two subgroups of patients with CRC with significantly different overall survival (
= 0.011 in validation cohort). Last, we found that a single ctDNA methylation marker, cg10673833, could yield high sensitivity (89.7%) and specificity (86.8%) for detection of CRC and precancerous lesions in a high-risk population of 1493 participants in a prospective cohort study. Together, our findings showed the value of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of CRC.
A Paleoproterozoic leucogranite has been identified petrologically as highly fractionated granite near Huili in the Jiaobei Terrane of the North China Craton. This granite occurs as a 3×10km pluton ...in the metamorphic Paleoproterozoic Jingshan Group and was dated at 1.86Ga using zircon LA-ICP-MS U-Pb ages. The Huili leucogranite can be subdivided into three groups: groups I and II have normal leucogranite abundances with 2.22–2.57wt.% Na2O, 6.10–6.92wt.% K2O, and 71.68–73.07wt.% SiO2 and group III has a remarkably high Na2O concentration of 6.45–7.23wt.%, a low K2O content of 0.82–1.05wt.%, and 74.21–74.79%wt.% SiO2. Moreover, group III leucogranite comprises slightly lower Th, U, and Sr contents and considerably lower K-feldspar compatible elements, such as Rb, Ba, Pb, and Cs than those of groups I & II. All of these features strongly indicate the K-feldspar fractionation during the magma evolution from groups I and II to group III, which is strongly supported by the Pb isotope analysis. Group III leucogranite has a notably high U/Pb ratio of 0.30 to 0.45 with a normal U content of 1.42 to 2.21ppm and very low Pb content of 4 to 5ppm, indicating a significant K-feldspar fractionation of the magma. Group III leucogranite has high radiogenic Pb isotopes (i.e., 206Pb/204Pb=37.407–116.31), demonstrating that the rocks must have existed in a high U-Pb system for a notably long time. Fortunately, the whole rock Pb isotope values roughly define the Pb-Pb isochrons, not only when all three groups of samples are considered together but also when any individual group is considered. The ages are very close to the formation time of ~1.86Ga indicating the high U/Pb system required by these Pb isotopes must have been initiated during the formation of the Huili leucogranite, which has perfectly confirmed the K-feldspar fractionation during the magma evolution.
All samples have consistent εNd values (t=1.86Ga) of −4.0 to −6.2 with two-stage Nd model ages (TDM2) of 2.67 to 2.85Ga. It is concluded that the 1.86Ga Huili leucogranite is highly fractionated, derived from ancient crustal material extracted from the depleted mantle at 2.7–2.8Ga, and experienced significant fractionation of K-feldspar in association with biotite and monazite fractionation during the magmatic evolution. Group III leucogranite is albite granite with albite phenocrysts, whereas groups I & II are K-feldspar granites with K-feldspar phenocrysts. It is here argued that the Huili albite granite (group III leucogranite) is derived from a highly differentiated residual melt separated from the K-rich magma represented by groups I and II leucogranite due to Kf fractionation during magma evolution.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Flavonoids, which modulate plant resistance to various stresses, can be induced by high light. B‐box (BBX) transcription factors (TFs) play crucial roles in the transcriptional regulation of ...flavonoids biosynthesis, but limited information is available on the association of BBX proteins with high light. We present a detailed overview of 45 Populus trichocarpa BBX TFs. Phylogenetic relationships, gene structure, tissue‐specific expression patterns and expression profiles were determined under 10 stress or phytohormone treatments to screen candidate BBX proteins associated with the flavonoid pathway. Sixteen candidate genes were identified, of which five were expressed predominantly in young leaves and roots, and BBX23 showed the most distinct response to high light. Overexpression of BBX23 in poplar activated expression of MYB TFs and structural genes in the flavonoid pathway, thereby promoting the accumulation of proanthocyanidins and anthocyanins. CRISPR/Cas9‐generated knockout of BBX23 resulted in the opposite trend. Furthermore, the phenotype induced by BBX23 overexpression was enhanced under exposure to high light. BBX23 was capable of binding directly to the promoters of proanthocyanidin‐ and anthocyanin‐specific genes, and its interaction with HY5 enhanced activation activity. We identified novel regulators of flavonoid biosynthesis in poplar, thereby enhancing our general understanding of the transcriptional regulatory mechanisms involved.
The BBX transcription factors PtrBBX23 promote anthocyanins and proanthocyanidins accumulation by directly regulating the expression of MYB transcription factors and structural genes in the flavonoid pathway in response to high light.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Upper gastrointestinal cancers (including oesophageal cancer and gastric cancer) are the most common cancers worldwide. Artificial intelligence platforms using deep learning algorithms have made ...remarkable progress in medical imaging but their application in upper gastrointestinal cancers has been limited. We aimed to develop and validate the Gastrointestinal Artificial Intelligence Diagnostic System (GRAIDS) for the diagnosis of upper gastrointestinal cancers through analysis of imaging data from clinical endoscopies.
This multicentre, case-control, diagnostic study was done in six hospitals of different tiers (ie, municipal, provincial, and national) in China. The images of consecutive participants, aged 18 years or older, who had not had a previous endoscopy were retrieved from all participating hospitals. All patients with upper gastrointestinal cancer lesions (including oesophageal cancer and gastric cancer) that were histologically proven malignancies were eligible for this study. Only images with standard white light were deemed eligible. The images from Sun Yat-sen University Cancer Center were randomly assigned (8:1:1) to the training and intrinsic verification datasets for developing GRAIDS, and the internal validation dataset for evaluating the performance of GRAIDS. Its diagnostic performance was evaluated using an internal and prospective validation set from Sun Yat-sen University Cancer Center (a national hospital) and additional external validation sets from five primary care hospitals. The performance of GRAIDS was also compared with endoscopists with three degrees of expertise: expert, competent, and trainee. The diagnostic accuracy, sensitivity, specificity, positive predictive value, and negative predictive value of GRAIDS and endoscopists for the identification of cancerous lesions were evaluated by calculating the 95% CIs using the Clopper-Pearson method.
1 036 496 endoscopy images from 84 424 individuals were used to develop and test GRAIDS. The diagnostic accuracy in identifying upper gastrointestinal cancers was 0·955 (95% CI 0·952–0·957) in the internal validation set, 0·927 (0·925–0·929) in the prospective set, and ranged from 0·915 (0·913–0·917) to 0·977 (0·977–0·978) in the five external validation sets. GRAIDS achieved diagnostic sensitivity similar to that of the expert endoscopist (0·942 95% CI 0·924–0·957 vs 0·945 0·927–0·959; p=0·692) and superior sensitivity compared with competent (0·858 0·832–0·880, p<0·0001) and trainee (0·722 0·691–0·752, p<0·0001) endoscopists. The positive predictive value was 0·814 (95% CI 0·788–0·838) for GRAIDS, 0·932 (0·913–0·948) for the expert endoscopist, 0·974 (0·960–0·984) for the competent endoscopist, and 0·824 (0·795–0·850) for the trainee endoscopist. The negative predictive value was 0·978 (95% CI 0·971–0·984) for GRAIDS, 0·980 (0·974–0·985) for the expert endoscopist, 0·951 (0·942–0·959) for the competent endoscopist, and 0·904 (0·893–0·916) for the trainee endoscopist.
GRAIDS achieved high diagnostic accuracy in detecting upper gastrointestinal cancers, with sensitivity similar to that of expert endoscopists and was superior to that of non-expert endoscopists. This system could assist community-based hospitals in improving their effectiveness in upper gastrointestinal cancer diagnoses.
The National Key R&D Program of China, the Natural Science Foundation of Guangdong Province, the Science and Technology Program of Guangdong, the Science and Technology Program of Guangzhou, and the Fundamental Research Funds for the Central Universities.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Unconventional shale reservoirs as the result of extremely low matrix permeability, higher potential gas productivity requires not only sufficient gas-in-place, but also a high concentration of ...brittle minerals (silica and/or carbonate) that is amenable to hydraulic fracturing. Shale lithofacies is primarily defined by mineral composition and organic matter richness, and its representation as a 3-D model has advantages in recognizing productive zones of shale-gas reservoirs, designing horizontal wells and stimulation strategy, and aiding in understanding depositional process of organic-rich shale. A challenging and key step is to effectively recognize shale lithofacies from well conventional logs, where the relationship is very complex and nonlinear. In the recognition of shale lithofacies, the application of support vector machine (SVM), which underlies statistical learning theory and structural risk minimization principle, is superior to the traditional empirical risk minimization principle employed by artificial neural network (ANN). We propose SVM classifier combined with learning algorithms, such as grid searching, genetic algorithm and particle swarm optimization, and various kernel functions the approach to identify Marcellus Shale lithofacies. Compared with ANN classifiers, the experimental results of SVM classifiers showed higher cross-validation accuracy, better stability and less computational time cost. The SVM classifier with radius basis function as kernel worked best as it is trained by particle swarm optimization. The lithofacies predicted using the SVM classifier are used to build a 3-D Marcellus Shale lithofacies model, which assists in identifying higher productive zones, especially with thermal maturity and natural fractures.
•Introduce SVM into shale lithofacies prediction.•Make lithofacies recognition more reliable and higher cross-validation accuracy.•Investigate various learning algorithms and kernel functions.•Build 3-D Marcellus Shale lithofacies model with the help of SVM.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
It has been shown that certain host minerals can robustly shield mineral inclusions and prevent loss of petrochronological information during subsequent thermal events. Rutile inclusions in garnet ...may crystallize under a wide range of metamorphic conditions and thus rutile is expected to be a useful geochronometer for constraining different stages of a metamorphic P-T path. Hence, it is important to ascertain whether rutile inclusions shielded by garnet retain the original crystallization age information despite later retrograde cooling or subsequent new thermal events. In this study, we test the “shielding effect” of garnet on rutile inclusions from the Baizhangya eclogite in the North Dabie Zone (China), which underwent a multi-stage thermal evolution. Secondary ion mass spectrometry (SIMS) UPb dating of inherited zircon core domains constrains a Neoproterozoic formation age (~770 Ma) of the eclogite protolith. SIMS UPb dating of metamorphic zircon and complementary thermal-ionization mass spectrometry (TIMS) SmNd isochron dating of garnet revealed Triassic eclogite-facies metamorphic ages (245–210 Ma). An Early Cretaceous SIMS UPb age of ca. 130 Ma, yielded by matrix rutile grains and rutile inclusions, suggests significant isotopic alteration since crystallization. This indicates that shielding was ineffective, since the UPb ages derived from the rutile inclusions, although hermetically armored by garnet, were influenced by a subsequent Early Cretaceous tectono-thermal event. Further transmission electron microscope (TEM) analysis revealed the presence of hematite microlites along the garnet-rutile boundaries, which may have acted as a potential reservoir to store the expelled radiogenic Pb from rutile. This study suggests that newly formed minerals located along mineral boundaries seem to be crucial for understanding the shielding effect.
•Zircon UPb and garnet SmNd dating constrains Triassic eclogite-facies metamorphism.•Matrix rutile and rutile inclusions in garnet yield consistent Early Cretaceous ages.•Garnet shielding on rutile inclusions under multi-thermal conditions is ineffective.•Nano-sized hematite microlites along the boundary act as a potential Pb sink.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Long noncoding RNAs (lncRNAs) have recently emerged as important regulators in governing fundamental biological processes, and many of which are likely to have functional roles in tumorigenesis. ...Maternally expressed gene 3 (MEG3) gene encodes a lncRNA whose expression is lost in an expanding list of primary human tumors and tumor cell lines, however its biological role and regulatory mechanism in gastric cancer (GC) development and progression are poorly defined.
Quantitative RT-PCR analysis was used to determine whether aberrant MEG3 expression was associated with GC patients pTNM stage and pM state. Furthermore, the effect of ectopic expression of MEG3 on cell proliferation, migration, invasion and cell apoptosis was assessed by using CCK-8, wound healing, transwell invasion assays and flow cytometric analysis, respectively, in GC cell lines HGC-27 and MGC-803. Moreover, the competing endogenous RNA (ceRNA) activity of MEG3 on miR-181a was investigated via luciferase reporter assay and immunoblot analysis.
MEG3 is decreased in GC patients and cell lines, and its expression was associated with metastatic GC. Furthermore, ectopic expression of MEG3 in HGC-27 and MGC-803 cells inhibited cell proliferation, migration, invasion, and promoted cell apoptosis, which might be due to MEG3 sequestering oncogenic miR-181 s in GC cells. Furthermore, MEG3 could up-regulated Bcl-2 via its competing endogenous RNA (ceRNA) activity on miR-181a.
These findings suggest that lncRNA MEG3, a ceRNA of miR-181 s, could regulate gastric carcinogenesis and may serve as a potential target for antineoplastic therapies.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK