Olfactory sensory experience regulates gliomagenesis via neuronal IGF1.

Animals constantly receive various sensory stimuli, such as odours, sounds, light and touch, from the surrounding environment. These sensory inputs are essential for animals to search for food and avoid predators, but they also affect their physiological status, and may cause diseases such as cancer. Malignant gliomas-the most lethal form of brain tumour1-are known to intimately communicate with neurons at the cellular level2,3. However, it remains unclear whether external sensory stimuli can directly affect the development of malignant glioma under normal living conditions. Here we show that olfaction can directly regulate gliomagenesis. In an autochthonous mouse model that recapitulates adult gliomagenesis4-6 originating in oligodendrocyte precursor cells (OPCs), gliomas preferentially emerge in the olfactory bulb-the first relay of brain olfactory circuitry. Manipulating the activity of olfactory receptor neurons (ORNs) affects the development of glioma. Mechanistically, olfaction excites mitral and tufted (M/T) cells, which receive sensory information from ORNs and release insulin-like growth factor 1 (IGF1) in an activity-dependent manner. Specific knockout of Igf1 in M/T cells suppresses gliomagenesis. In addition, knocking out the IGF1 receptor in pre-cancerous mutant OPCs abolishes the ORN-activity-dependent mitogenic effects. Our findings establish a link between sensory experience and gliomagenesis through their corresponding sensory neuronal circuits.

Deubiquitination of CDC6 by OTUD6A promotes tumour progression and chemoresistance.

BACKGROUND: CDC6 is an oncogenic protein whose expression level fluctuates during the cell cycle. Although several E3 ubiquitin ligases responsible for the ubiquitin-mediated proteolysis of CDC6 have been identified, the deubiquitination pathway for CDC6 has not been investigated. METHODS: The proteome-wide deubiquitinase (DUB) screening was used to identify the potential regulator of CDC6. Immunofluorescence, protein half-life and deubiquitination assays were performed to determine the protein stability of CDC6. Gain- and loss-of-function experiments were implemented to analyse the impacts of OUTD6A-CDC6 axis on tumour growth and chemosensitivity in vitro. N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN)-induced conditional Otud6a knockout (CKO) mouse model and tumour xenograft model were performed to analyse the role of OTUD6A-CDC6 axis in vivo. Tissue specimens were used to determine the association between OTUD6A and CDC6. RESULTS: OTUD6A interacts with, depolyubiquitinates and stabilizes CDC6 by removing K6-, K33-, and K48-linked polyubiquitination. Moreover, OTUD6A promotes cell proliferation and decreases sensitivity to chemotherapy by upregulating CDC6. CKO mice are less prone to BCa tumorigenesis induced by BBN, and knockdown of OTUD6A inhibits tumour progression in vivo. Furthermore, OTUD6A protein level has a positive correlation with CDC6 protein level, and high protein levels of OTUD6A and CDC6 are associated with poor prognosis in patients with bladder cancer. CONCLUSIONS: We reveal an important yet missing piece of novel DUB governing CDC6 stability. In addition, our findings propose a model for the OTUD6A-CDC6 axis that provides novel insights into cell cycle and chemosensitivity regulation, which may become a potential biomarker and promising drug target for cancer treatment.

MdGH3.6 is targeted by MdMYB94 and plays a negative role in apple water-deficit stress tolerance.

Drought significantly limits apple fruit production and quality. Decoding the key genes involved in drought stress tolerance is important for breeding varieties with improved drought resistance. Here, we identified GRETCHEN HAGEN3.6 (GH3.6), an indole-3-acetic acid (IAA) conjugating enzyme, to be a negative regulator of water-deficit stress tolerance in apple. Overexpressing MdGH3.6 reduced IAA content, adventitious root number, root length and water-deficit stress tolerance, whereas knocking down MdGH3.6 and its close paralogs increased IAA content, adventitious root number, root length and water-deficit stress tolerance. Moreover, MdGH3.6 negatively regulated the expression of wax biosynthetic genes under water-deficit stress and thus negatively regulated cuticular wax content. Additionally, MdGH3.6 negatively regulated reactive oxygen species scavengers, including antioxidant enzymes and metabolites involved in the phenylpropanoid and flavonoid pathway in response to water-deficit stress. Further study revealed that the homolog of transcription factor AtMYB94, rather than AtMYB96, could bind to the MdGH3.6 promoter and negatively regulated its expression under water-deficit stress conditions in apple. Overall, our results identify a candidate gene for the improvement of drought resistance in fruit trees.

Zinc-finger protein MdBBX7/MdCOL9, a target of MdMIEL1 E3 ligase, confers drought tolerance in apple.

Water deficit is one of the main challenges for apple (Malus × domestica) growth and productivity. Breeding drought-tolerant cultivars depends on a thorough understanding of the drought responses of apple trees. Here, we identified the zinc-finger protein B-BOX 7/CONSTANS-LIKE 9 (MdBBX7/MdCOL9), which plays a positive role in apple drought tolerance. The overexpression of MdBBX7 enhanced drought tolerance, whereas knocking down MdBBX7 expression reduced it. Chromatin immunoprecipitation-sequencing (ChIP-seq) analysis identified one cis-element of MdBBX7, CCTTG, as well as its known binding motif, the T/G box. ChIP-seq and RNA-seq identified 1,197 direct targets of MdBBX7, including ETHYLENE RESPONSE FACTOR (ERF1), EARLY RESPONSIVE TO DEHYDRATION 15 (ERD15), and GOLDEN2-LIKE 1 (GLK1) and these were further verified by ChIP-qPCR and electronic mobility shift assays. Yeast two-hybrid screen identified an interacting protein of MdBBX7, RING-type E3 ligase MYB30-INTERACTING E3 LIGASE 1 (MIEL1). Further examination revealed that MdMIEL1 could mediate the ubiquitination and degradation of MdBBX7 by the 26S proteasome pathway. Genetic interaction analysis suggested that MdMIEL1 acts as an upstream factor of MdBBX7. In addition, MdMIEL1 was a negative regulator of the apple drought stress response. Taken together, our results illustrate the molecular mechanisms by which the MdMIEL1-MdBBX7 module influences the response of apple to drought stress.

The positive feedback regulatory loop of miR160-Auxin Response Factor 17-HYPONASTIC LEAVES 1 mediates drought tolerance in apple trees.

Drought stress tolerance is a complex trait regulated by multiple factors. Here, we demonstrate that the miRNA160-Auxin Response Factor 17 (ARF17)-HYPONASTIC LEAVES 1 module is crucial for apple (Malus domestica) drought tolerance. Using stable transgenic plants, we found that drought tolerance was improved by higher levels of Mdm-miR160 or MdHYL1 and by decreased levels of MdARF17, whereas reductions in MdHYL1 or increases in MdARF17 led to greater drought sensitivity. Further study revealed that modulation of drought tolerance was achieved through regulation of drought-responsive miRNA levels by MdARF17 and MdHYL1; MdARF17 interacted with MdHYL1 and bound to the promoter of MdHYL1. Genetic analysis further suggested that MdHYL1 is a direct downstream target of MdARF17. Importantly, MdARF17 and MdHYL1 regulated the abundance of Mdm-miR160. In addition, the Mdm-miR160-MdARF17-MdHYL1 module regulated adventitious root development. We also found that Mdm-miR160 can move from the scion to the rootstock in apple and tomato (Solanum lycopersicum), thereby improving root development and drought tolerance of the rootstock. Our study revealed the mechanisms by which the positive feedback loop of Mdm-miR160-MdARF17-MdHYL1 influences apple drought tolerance.

Pyroptosis predicts immunotherapy outcomes across multiple cancer types.

Pyroptosis is a programmed cell death characterized by inflammation and may coordinate with cancer immunotherapy, but assessments of pyroptosis in patients with immunotherapy are lacking. We evaluated the pyroptosis potentials in 71 cohorts with 24,388 cancer patients. They were elevated in tumors compared to normal tissues but had a weak relationship with prognosis. High pyroptosis potentials indicated "hot tumors" characteristics and high objective response rates to PD1/PDL1 inhibitors derived from clinical trials. In 15 cohorts with patients treated with immunotherapy, a pyroptosis score showed predictive values in objective response rate, progression-free survival, and overall survival. Systematic treatments, such as chemotherapy or endocrine therapy, enhanced pyroptosis in drug-resistant tumors. These results were further validated in three independent clinical cohorts and our two institutional cohorts by immunohistochemistry. Our findings uncover a value of pyroptosis potentials to predict immunotherapy responses and a theoretical rationale for combining pyroptosis inducers and immunotherapy in cancer treatment.

Fine-tuning of SUMOylation modulates drought tolerance of apple.

SUMOylation is involved in various aspects of plant biology, including drought stress. However, the relationship between SUMOylation and drought stress tolerance is complex; whether SUMOylation has a crosstalk with ubiquitination in response to drought stress remains largely unclear. In this study, we found that both increased and decreased SUMOylation led to increased survival of apple (Malus × domestica) under drought stress: both transgenic MdSUMO2A overexpressing (OE) plants and MdSUMO2 RNAi plants exhibited enhanced drought tolerance. We further confirmed that MdDREB2A is one of the MdSUMO2 targets. Both transgenic MdDREB2A OE and MdDREB2AK192R OE plants (which lacked the key site of SUMOylation by MdSUMO2A) were more drought tolerant than wild-type plants. However, MdDREB2AK192R OE plants had a much higher survival rate than MdDREB2A OE plants. We further showed SUMOylated MdDREB2A was conjugated with ubiquitin by MdRNF4 under drought stress, thereby triggering its protein degradation. In addition, MdRNF4 RNAi plants were more tolerant to drought stress. These results revealed the molecular mechanisms that underlie the relationship of SUMOylation with drought tolerance and provided evidence for the tight control of MdDREB2A accumulation under drought stress mediated by SUMOylation and ubiquitination.

MdZAT5 regulates drought tolerance via mediating accumulation of drought-responsive miRNAs and mRNAs in apple.

Drought limits apple yield and fruit quality. However, the molecular mechanism of apple in response to drought is not well known. Here, we report a Cys2/His2 (C2H2)-type zinc-finger protein, MdZAT5, that positively regulates apple drought tolerance by regulating drought-responsive RNAs and microRNAs (miRNAs). DNA affinity purification and sequencing and yeast-one hybrid analysis identified the binding motifs of MdZAT5, T/ACACT/AC/A/G. Chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) and electrophoretic mobility shift assays (EMSAs) showed that MdZAT5 directly binds to the promoters of the drought-responsive genes including MdRHA2a, MdLEA14, MdTPX1, and MdCAT3, and activates their expression under drought stress. MdZAT5 interacts with and directly targets HYPONASTIC LEAVES1 (MdHYL1). MdZAT5 may facilitate the interaction of MdHYL1 with pri-miRNAs or MdDCL1 by activating MdHYL1 expression, thereby regulating the biogenesis of drought-responsive miRNAs. Genetic dissection showed that MdHYL1 is essential for MdZAT5-mediated drought tolerance and miRNA biogenesis. In addition, ChIP-qPCR and EMSA revealed that MdZAT5 binds directly to the promoters of some MIR genes including Mdm-miR171i and Mdm-miR172c, and modulates their transcription. Taken together, our findings improve our understanding of the molecular mechanisms of drought response in apple and provide a candidate gene for the breeding of drought-tolerant cultivars.

A multifaceted module of BRI1 ETHYLMETHANE SULFONATE SUPRESSOR1 (BES1)-MYB88 in growth and stress tolerance of apple.

The R2R3 transcription factor MdMYB88 has previously been reported to function in biotic and abiotic stress responses. Here, we identify BRI1 ETHYLMETHANE SULFONATE SUPRESSOR1 (MdBES1), a vital component of brassinosteroid (BR) signaling in apple (Malus × domestica) that directly binds to the MdMYB88 promoter, regulating the expression of MdMYB88 in a dynamic and multifaceted mode. MdBES1 positively regulated expression of MdMYB88 under cold stress and pathogen attack, but negatively regulated its expression under control and drought conditions. Consistently, MdBES1 was a positive regulator for cold tolerance and disease resistance in apple, but a negative regulator for drought tolerance. In addition, MdMYB88 participated in BR biosynthesis by directly regulating the BR biosynthetic genes DE ETIOLATED 2 (MdDET2), DWARF 4 (MdDWF4), and BRASSINOSTEROID 6 OXIDASE 2 (MdBR6OX2). Applying exogenous BR partially rescued the erect leaf and dwarf phenotypes, as well as defects in stress tolerance in MdMYB88/124 RNAi plants. Moreover, knockdown of MdMYB88 in MdBES1 overexpression (OE) plants decreased resistance to a pathogen and C-REPEAT BINDING FACTOR1 expression, whereas overexpressing MdMYB88 in MdBES1 OE plants increased expression of SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 （MdSPL3） and BR biosynthetic genes, suggesting that MdMYB88 contributes to MdBES1 function during BR biosynthesis and the stress response. Taken together, our results reveal multifaceted regulation of MdBES1 on MdMYB88 in BR biosynthesis and stress tolerance.

Insights into the effect of human civilization on Malus evolution and domestication.

The evolutionary history of the Malus genus has not been well studied. In the current study, we presented genetic evidence on the origin of the Malus genus based on genome sequencing of 297 Malus accessions, revealing the genetic relationship between wild species and cultivated apples. Our results demonstrated that North American and East Asian wild species are closer to the outgroup (pear) than Central Asian species, and hybrid species including natural (separated before the Pleistocene, about 2.5 Mya) and artificial hybrids (including ornamental trees and rootstocks) are between East and Central Asian wild species. Introgressions from M. sylvestris in cultivated apples appeared to be more extensive than those from M. sieversii, whose genetic background flowed westward across Eurasia and eastward to wild species including M. prunifolia, M. × asiatica, M. × micromalus, and M. × robust. Our results suggested that the loss of ancestral gene flow from M. sieversii in cultivated apples accompanied the movement of European traders around the world since the Age of Discovery. Natural SNP variations showed that cultivated apples had higher nucleotide diversity than wild species and more unique SNPs than other apple groups. An apple ERECTA-like gene that underwent selection during domestication on 15th chromosome was identified as a likely major determinant of fruit length and diameter, and an NB-ARC domain-containing gene was found to strongly affect anthocyanin accumulation using a genome-wide association approach. Our results provide new insights into the origin and domestication of apples and will be useful in new breeding programmes and efforts to increase fruit crop productivity.

Abscisic acid homeostasis is mediated by feedback regulation of MdMYB88 and MdMYB124.

The phytohormone abscisic acid (ABA) is involved in various plant processes. In response to drought stress, plants quickly accumulate ABA, but the regulatory mechanism of ABA accumulation is largely unknown, especially in woody plants. In this study, we report that MdMYB88 and MdMYB124 are myeloblastosis (MYB) transcription factors critical for ABA accumulation in apple trees (Malus x domestica) following drought, and this regulation is negatively controlled by ABA. MdMYB88 and MdMYB124 positively regulate leaf water transpiration, photosynthetic capacity, and stress endurance in apple trees under drought conditions. MdMYB88 and MdMYB124 regulate the expression of biosynthetic and catabolic genes of ABA, as well as drought- and ABA- responsive genes. MdMYB88 associates with promoter regions of the ABA biosynthetic gene 9-cis-epoxycarotenoid dioxygenase 3 (NCED3). Finally, expression of MdMYB88 and MdMYB124 is repressed by ABA. Our results identify a feedback regulation of MdMYB88 and MdMYB124 in modulating ABA homeostasis in apple trees.

Terthiophene-functionalized mesoporous silica-based fluorescence sensor for the detection of trace methyl orange in aqueous media.

A terthiophene-functionalized mesoporous SBA-15 silica, i.e., TTU-SBA-15, was successfully developed and used as a highly selective and ultrasensitive fluorescence sensor for methyl orange (MO) detection. When the concentration of MO was increased, the fluorescence emission intensity of TTU-SBA-15 suspensions at 452 nm gradually decreased at an excitation wavelength of 368 nm, and the color of the suspension solutions changed obviously from blue to dark under 365 nm UV light. The fluorescence intensity at 452 nm was linearly proportional to the concentration of MO in the range 0.20 - 2.0 µM, with a detection limit of 0.092 µM. Competitive pollutants, variations in pH, and sample recycling had subtle or negligible effects on the detection of MO. TTU-SBA-15 was applied to the determination of MO in tap water, and recoveries from spiked samples were in the range 98.3 - 103.0%. This study provides a convenient and effective strategy to realize highly sensitive and selective sensors that could target dyes via the functional modification of mesoporous materials.

Thymine DNA glycosylase-regulated TAZ promotes radioresistance by targeting nonhomologous end joining and tumor progression in esophageal cancer.

Radiation resistance is a major cause of esophageal cancer relapse or metastasis. Transcriptional coactivator with PDZ binding domain (TAZ) is a final effector of the Hippo signaling pathway and plays critical roles in several types of cancer, but how it participates in the progression and radiation resistance of esophageal cancer remains unclear. Here, we revealed that TAZ was the strongest prognostic factor among Hippo pathway members. Overexpression of TAZ predicted poor outcome and adverse pathological features. In cell and animal models, TAZ facilitated cell proliferation, motility, and radiation resistance. Additionally, TAZ promoted expression of nonhomologous end joining (NHEJ)-related genes, which are the main contributors to repair irradiation-induced DNA breaks and result in radiation resistance. Amplification of the TAZ gene occurred in 2.5%-3.2% of esophageal cancers. In addition, the CpG islands of the TAZ gene were demethylated in esophageal cancer under thymine DNA glycosylase (TDG) regulation. Knockdown of TDG inhibited cell growth, motility, and radiation resistance, which were overridden by TAZ overexpression. Collectively, these findings suggest that the TDG/TAZ/NHEJ axis is a critical player in esophageal cancer progression and radiation resistance, as well as a potential target for radiotherapy.

GSTU43 gene involved in ALA-regulated redox homeostasis, to maintain coordinated chlorophyll synthesis of tomato at low temperature.

BACKGROUND: Exogenous 5-aminolevulinic acid (ALA) positively regulates plants chlorophyll synthesis and protects them against environmental stresses, although the protection mechanism is not fully clear. Here, we explored the effects of ALA on chlorophyll synthesis in tomato plants, which are sensitive to low temperature. We also examined the roles of the glutathione S-transferase (GSTU43) gene, which is involved in ALA-induced tolerance to oxidation stress and regulation of chlorophyll synthesis under low temperature. RESULTS: Exogenous ALA alleviated low temperature caused chlorophyll synthesis obstacle of uroporphyrinogen III (UROIII) conversion to protoporphyrin IX (Proto IX), and enhanced the production of chlorophyll and its precursors, including endogenous ALA, Proto IX, Mg-protoporphyrin IX (Mg-proto IX), and protochlorophyll (Pchl), under low temperature in tomato leaves. However, ALA did not regulate chlorophyll synthesis at the level of transcription. Notably, ALA up-regulated the GSTU43 gene and protein expression and increased GST activity. Silencing of GSTU43 with virus-induced gene silencing reduced the activities of GST, superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase, and increased the membrane lipid peroxidation; while fed with ALA significant increased all these antioxidase activities and antioxidant contents, and alleviated the membrane damage. CONCLUSIONS: ALA triggered GST activity encoded by GSTU43, and increased tomato tolerance to low temperature-induced oxidative stress, perhaps with the assistance of ascorbate- and/or a glutathione-regenerating cycles, and actively regulated the plant redox homeostasis. This latter effect reduced the degree of membrane lipid peroxidation, which was essential for the coordinated synthesis of chlorophyll.

MdMYB88 and MdMYB124 Enhance Drought Tolerance by Modulating Root Vessels and Cell Walls in Apple.

Water deficit is one of the main limiting factors in apple (Malus × domestica Borkh.) cultivation. Root architecture plays an important role in the drought tolerance of plants; however, research efforts to improve drought tolerance of apple trees have focused on aboveground targets. Due to the difficulties associated with visualization and data analysis, there is currently a poor understanding of the genetic players and molecular mechanisms involved in the root architecture of apple trees under drought conditions. We previously observed that MdMYB88 and its paralog MdMYB124 regulate apple tree root morphology. In this study, we found that MdMYB88 and MdMYB124 play important roles in maintaining root hydraulic conductivity under long-term drought conditions and therefore contribute toward adaptive drought tolerance. Further investigation revealed that MdMYB88 and MdMYB124 regulate root xylem development by directly binding MdVND6 and MdMYB46 promoters and thus influence expression of their target genes under drought conditions. In addition, MdMYB88 and MdMYB124 were shown to regulate the deposition of cellulose and lignin root cell walls in response to drought. Taken together, our results provide novel insights into the importance of MdMYB88 and MdMYB124 in root architecture, root xylem development, and secondary cell wall deposition in response to drought in apple trees.

miR-146a-5p mediates epithelial-mesenchymal transition of oesophageal squamous cell carcinoma via targeting Notch2.

This corrects the article DOI: 10.1038/bjc.2016.367.

An atypical R2R3 MYB transcription factor increases cold hardiness by CBF-dependent and CBF-independent pathways in apple.

Apple (Malus × domestica) trees are vulnerable to freezing temperatures. However, there has been only limited success in developing cold-hardy cultivars. This lack of progress is due at least partly to lack of understanding of the molecular mechanisms of freezing tolerance in apple. In this study, we evaluated the potential roles for two R2R3 MYB transcription factors (TFs), MYB88 and the paralogous FLP (MYB124), in cold stress in apple and Arabidopsis. We found that MYB88 and MYB124 positively regulate freezing tolerance and cold-responsive gene expression in both apple and Arabidopsis. Chromatin-Immunoprecipitation-qPCR and electrophoretic mobility shift assays showed that MdMYB88/MdMYB124 act as direct regulators of the COLD SHOCK DOMAIN PROTEIN 3 (MdCSP3) and CIRCADIAN CLOCK ASSOCIATED 1 (MdCCA1) genes. Dual luciferase reporter assay indicated that MdCCA1 but not MdCSP3 activated the expression of MdCBF3 under cold stress. Moreover, MdMYB88 and MdMYB124 promoted anthocyanin accumulation and H2 O2 detoxification in response to cold. Taken together, our results suggest that MdMYB88 and MdMYB124 positively regulate cold hardiness and cold-responsive gene expression under cold stress by C-REPEAT BINDING FACTOR (CBF)-dependent and CBF-independent pathways.

RACK1 promotes radiation resistance in esophageal cancer via regulating AKT pathway and Bcl-2 expression.

RACK1 is a seven Trp-Asp 40 repeat protein, which interacts with a wide range of kinases and proteins. RACK1 plays an important role in the proliferation and progression of various cancers. The aim of this study is to detect the role of RACK1 in the radioresistance in esophageal cancer. The results indicated that downregulation of RACK1 reduced the colony formation ability, proliferation ability and resistance of cells to radiation effection through regulating the radiation-related proteins including pAKT, Bcl-2 and Bim; whereas upregulation of RACK1 promoted the ability and radioresistance of ESCC cells. Our findings suggest that RACK1 promotes proliferation and radioresistance in ESCC cells by activating the AKT pathway, upregulating Bcl-2 expression and downregulating protein levels of Bim. Our study fills in gaps in the field of RACK1 and radiation resistance and may provide new possibilities for improving strategies of radiotherapy in esophageal cancer.

miR-146a-5p mediates epithelial-mesenchymal transition of oesophageal squamous cell carcinoma via targeting Notch2.

BACKGROUND: Our previous study found that dysregulated microRNA-146a-5p (miR-146a-5p) is involved in oesophageal squamous cell cancer (ESCC) proliferation. This article aimed to evaluate its detailed mechanisms in ESCC epithelial-mesenchymal transition (EMT) progression. METHODS: Invasion assay, qRT-PCR and western blotting were used to validate the roles of miR-146a-5p and Notch2 in EMT progression. miRNA target gene prediction databases and dual-luciferase reporter assay were used to validate the target gene. RESULTS: miR-146a-5p inhibitor led to increase of invaded ESCC cells, while miR-146a-5p mimics inhibited invasion ability of ESCC cells. Protein level of E-cadherin decreased, whereas those of Snail and Vimentin increased in the anti-miR-146a-5p group, which demonstrated that miR-146a-5p inhibits EMT progression of ESCC cells. miRNA target gene prediction databases indicated the potential of Notch2 as a direct target gene of miR-146a-5p and dual-luciferase reporter assay validated it. Importantly, shRNA-Notch2 restrained EMT and partially abrogated the inhibiting effects of miR-146a-5p on EMT progression of ESCC cells. CONCLUSIONS: miR-146a-5p functions as a tumour-suppressive miRNA targeting Notch2 and inhibits the EMT progression of ESCC.