In vivo single-cell CRISPR uncovers distinct TNF programmes in tumour evolution.

The tumour evolution model posits that malignant transformation is preceded by randomly distributed driver mutations in cancer genes, which cause clonal expansions in phenotypically normal tissues. Although clonal expansions can remodel entire tissues1-3, the mechanisms that result in only a small number of clones transforming into malignant tumours remain unknown. Here we develop an in vivo single-cell CRISPR strategy to systematically investigate tissue-wide clonal dynamics of the 150 most frequently mutated squamous cell carcinoma genes. We couple ultrasound-guided in utero lentiviral microinjections, single-cell RNA sequencing and guide capture to longitudinally monitor clonal expansions and document their underlying gene programmes at single-cell transcriptomic resolution. We uncover a tumour necrosis factor (TNF) signalling module, which is dependent on TNF receptor 1 and involving macrophages, that acts as a generalizable driver of clonal expansions in epithelial tissues. Conversely, during tumorigenesis, the TNF signalling module is downregulated. Instead, we identify a subpopulation of invasive cancer cells that switch to an autocrine TNF gene programme associated with epithelial-mesenchymal transition. Finally, we provide in vivo evidence that the autocrine TNF gene programme is sufficient to mediate invasive properties and show that the TNF signature correlates with shorter overall survival of patients with squamous cell carcinoma. Collectively, our study demonstrates the power of applying in vivo single-cell CRISPR screening to mammalian tissues, unveils distinct TNF programmes in tumour evolution and highlights the importance of understanding the relationship between clonal expansions in epithelia and tumorigenesis.

Control of dynamic cell behaviors during angiogenesis and anastomosis by Rasip1.

Organ morphogenesis is driven by a wealth of tightly orchestrated cellular behaviors, which ensure proper organ assembly and function. Many of these cell activities involve cell-cell interactions and remodeling of the F-actin cytoskeleton. Here, we analyze the requirement for Rasip1 (Ras-interacting protein 1), an endothelial-specific regulator of junctional dynamics, during blood vessel formation. Phenotype analysis of rasip1 mutants in zebrafish embryos reveals distinct functions of Rasip1 during sprouting angiogenesis, anastomosis and lumen formation. During angiogenic sprouting, loss of Rasip1 causes cell pairing defects due to a destabilization of tricellular junctions, indicating that stable tricellular junctions are essential to maintain multicellular organization within the sprout. During anastomosis, Rasip1 is required to establish a stable apical membrane compartment; rasip1 mutants display ectopic, reticulated junctions and the apical compartment is frequently collapsed. Loss of Ccm1 and Heg1 function mimics the junctional defects of rasip1 mutants. Furthermore, downregulation of ccm1 and heg1 leads to a delocalization of Rasip1 at cell junctions, indicating that junctional tethering of Rasip1 is required for its function in junction formation and stabilization during sprouting angiogenesis.

A massively parallel reporter assay reveals focused and broadly encoded RNA localization signals in neurons.

Asymmetric subcellular mRNA localization allows spatial regulation of gene expression and functional compartmentalization. In neurons, localization of specific mRNAs to neurites is essential for cellular functioning. However, it is largely unknown how transcript sorting works in a sequence-specific manner. Here, we combined subcellular transcriptomics and massively parallel reporter assays and tested ∼50 000 sequences for their ability to localize to neurites. Mapping the localization potential of >300 genes revealed two ways neurite targeting can be achieved: focused localization motifs and broadly encoded localization potential. We characterized the interplay between RNA stability and localization and identified motifs able to bias localization towards neurite or soma as well as the trans-acting factors required for their action. Based on our data, we devised machine learning models that were able to predict the localization behavior of novel reporter sequences. Testing this predictor on native mRNA sequencing data showed good agreement between predicted and observed localization potential, suggesting that the rules uncovered by our MPRA also apply to the localization of native full-length transcripts.

A Fully Automated Visual Grading System for White Matter Hyperintensities of T2-Fluid Attenuated Inversion Recovery Magnetic Resonance Imaging.

BACKGROUND: The Fazekas scale is one of the most commonly used visual grading systems for white matter hyperintensity (WMH) for brain disorders like dementia from T2-fluid attenuated inversion recovery magnetic resonance (MR) images (T2-FLAIRs). However, the visual grading of the Fazekas scale suffers from low-intra and inter-rater reliability and high labor-intensive work. Therefore, we developed a fully automated visual grading system using quantifiable measurements. METHODS: Our approach involves four stages: (1) the deep learning-based segmentation of ventricles and WMH lesions, (2) the categorization into periventricular white matter hyperintensity (PWMH) and deep white matter hyperintensity (DWMH), (3) the WMH diameter measurement, and (4) automated scoring, following the quantifiable method modified for Fazekas grading. We compared the performances of our method and that of the modified Fazekas scale graded by three neuroradiologists for 404 subjects with T2-FLAIR utilized from a clinical site in Korea. RESULTS: The Krippendorff's alpha across our method and raters (A) versus those only between the radiologists (R) were comparable, showing substantial (0.694 vs. 0.732; 0.658 vs. 0.671) and moderate (0.579 vs. 0.586) level of agreements for the modified Fazekas, the DWMH, and the PWMH scales, respectively. Also, the average of areas under the receiver operating characteristic curve between the radiologists (0.80 ± 0.09) and the radiologists against our approach (0.80 ± 0.03) was comparable. CONCLUSIONS: Our fully automated visual grading system for WMH demonstrated comparable performance to the radiologists, which we believe has the potential to assist the radiologist in clinical findings with unbiased and consistent scoring.

Reproducible imaging-based prediction of molecular subtype and risk stratification of gliomas across different experience levels using a structured reporting system.

OBJECTIVES: To determine reproducible MRI parameters predictive of molecular subtype and risk stratification in glioma and develop a structured reporting system. METHODS: All study patients were initially diagnosed with glioma, 141 from the Cancer Genome Atlas and 131 from our tertiary institution, as training and validation sets, respectively. Images were analyzed by three neuroradiologists with 1-7 years of experience. MRI features including contrast enhancement pattern, necrosis, margin, edema, T2/FLAIR mismatch, internal cyst, and cerebral blood volume higher than normal cortex were reported using a structured reporting system. The pathology was stratified into five risk types: (1) oligodendroglioma, isocitrate dehydrogenase [IDH]-mutant, 1p19q co-deleted; (2) diffuse astrocytoma, IDH-mutant, grade II-III; (3) glioblastoma, IDH-mutant, grade IV; (4) diffuse astrocytoma, IDH-wild, grade II-III; and (5) glioblastoma, IDH-wild, grade IV. Significant predictors were selected using multivariate logistic regression, and diagnostic performance was tested using a validation set. RESULTS: Reproducible imaging parameters exhibiting > 50% agreement across readers included the presence of necrosis, T2/FLAIR mismatch, internal cyst, and predominant contrast enhancement. In the validation set, prediction of risk type 5 exhibited the highest diagnostic performance with AUCs of 0.92 (reader 1) and 0.93 (reader 2) with predominant enhancement, followed by risk type 2 with AUCs of 0.95 and 0.95 with T2/FLAIR mismatch sign and no necrosis, and risk type 1 with AUCs of 0.84 and 0.83 with internal cyst or necrosis. Risk types 3 and 4 were difficult to visually predict. CONCLUSIONS: Imaging parameters with high reproducibility enabling prediction of IDH-wild-type glioblastoma, IDH-mutant/1p19q co-deletion oligodendroglioma, and IDH-mutant diffuse astrocytoma were identified. KEY POINTS: • Reproducible MRI parameters for determining molecular subtypes of glioma included the presence of necrosis, T2/FLAIR mismatch, internal cyst, and predominant contrast enhancement. • IDH-wild type glioblastoma, IDH-mutant/1p19q co-deletion oligodendroglioma, and IDH-mutant low-grade astrocytoma were identified using MRI parameters with high inter-reader reproducibility. • Identification of IDH-wild type low-grade glioma and IDH-mutant glioblastoma was difficult by visual analysis.

The Estimated Prevalence and Incidence of Endometriosis With the Korean National Health Insurance Service-National Sample Cohort (NHIS-NSC): A National Population-Based Study.

BACKGROUND: The incidence and prevalence of endometriosis remain unclear due to diagnostic difficulties. Especially, there has been little information regarding the population-based epidemiology of endometriosis. The purpose of this study is to estimate the prevalence and incidence of endometriosis in Korea based on the health insurance claims data. METHODS: This study is a retrospective cohort study using the Korean National Health Insurance Service-National Sample Cohort, which correspond to approximately 1 million Korean populations from 2002 to 2013. Patients aged 15-54 years were selected, and the prevalence and incidence of endometriosis were estimated by time and age groups. RESULTS: The age-adjusted prevalence rate of endometriosis also increased from 2.12 per 1,000 persons (95% confidence interval [CI], 2.01-2.24) in 2002 to 3.56 per 1,000 persons (95% CI, 3.40-3.71) in 2013. The average adjusted incidence showed no statistically significant increase. However, the age-specific incidence of the 15-19 and 20-24 years age groups increased significantly from 0.24 and 1.29 per 1,000 persons in 2003 to 2.73 and 2.71 per 1,000 persons in 2013 (R2 = 0.93 and 0.77, P < 0.001), while the incidence rate of the age group 40-44 and 45-49 years decreased from 2.36 and 1.72 per 1,000 persons in 2003 to 0.81 and 0.27 per 1,000 persons in 2013 (R2 = 0.83 and 0.89, P < 0.001). CONCLUSION: The prevalence and incidence of endometriosis in Korean women were lower than that of previous reports in high-risk population studies. Furthermore, we found a significant increase in the diagnosis of endometriosis in younger age groups.

Roles of Mis18α in epigenetic regulation of centromeric chromatin and CENP-A loading.

The Mis18 complex has been identified as a critical factor for the centromeric localization of a histone H3 variant, centromeric protein A (CENP-A), which is responsible for the specification of centromere identity in the chromosome. However, the functional role of Mis18 complex is largely unknown. Here, we generated Mis18α conditional knockout mice and found that Mis18α deficiency resulted in lethality at early embryonic stage with severe defects in chromosome segregation caused by mislocalization of CENP-A. Further, we demonstrate Mis18α's crucial role for epigenetic regulation of centromeric chromatin by reinforcing centromeric localization of DNMT3A/3B. Mis18α interacts with DNMT3A/3B, and this interaction is critical for maintaining DNA methylation and hence regulating epigenetic states of centromeric chromatin. Mis18α deficiency led to reduced DNA methylation, altered histone modifications, and uncontrolled noncoding transcripts in centromere region by decreased DNMT3A/3B enrichment. Together, our findings uncover the functional mechanism of Mis18α and its pivotal role in mammalian cell cycle.

EZH2 generates a methyl degron that is recognized by the DCAF1/DDB1/CUL4 E3 ubiquitin ligase complex.

Ubiquitination plays a major role in protein degradation. Although phosphorylation-dependent ubiquitination is well known for the regulation of protein stability, methylation-dependent ubiquitination machinery has not been characterized. Here, we provide evidence that methylation-dependent ubiquitination is carried out by damage-specific DNA binding protein 1 (DDB1)/cullin4 (CUL4) E3 ubiquitin ligase complex and a DDB1-CUL4-associated factor 1 (DCAF1) adaptor, which recognizes monomethylated substrates. Molecular modeling and binding affinity studies reveal that the putative chromo domain of DCAF1 directly recognizes monomethylated substrates, whereas critical binding pocket mutations of the DCAF1 chromo domain ablated the binding from the monomethylated substrates. Further, we discovered that enhancer of zeste homolog 2 (EZH2) methyltransferase has distinct substrate specificities for histone H3K27 and nonhistones exemplified by an orphan nuclear receptor, RORα. We propose that EZH2-DCAF1/DDB1/CUL4 represents a previously unrecognized methylation-dependent ubiquitination machinery specifically recognizing "methyl degron"; through this, nonhistone protein stability can be dynamically regulated in a methylation-dependent manner.

Analysis of MED12 Mutation in Multiple Uterine Leiomyomas in South Korean patients.

Uterine leiomyomas are one of the most common benign gynecologic tumors, but the exact causes are not completely understood. In 2011, through DNA sequencing, MED12 mutation was discovered in approximately 71% of uterine leiomyomas. Several recent studies confirmed the high frequency of MED12 mutation in uterine leiomyoma. Nevertheless, no study has been done on MED12 mutation in the case of patients with multiple leiomyomas in a patient. The purpose of this study was to investigate the frequency of MED12 mutations in uterine leiomyomas of South Korean patients. In addition, we examined MED12 mutation in multiple leiomyomas in the same patients. Uterine leiomyoma tissues were obtained from symptomatic women who underwent hysterectomy or myomectomy for medically indicated reasons. We collected 60 uterine leiomyomas from 41 women. Tumor size ranged from 1 to 12cm. Patients' ages ranged from 25 to 55 years with an average of 38.4 years. Of the 60 tumors, 40 (66.67%) displayed MED12 mutation. Among the 41 patients, 14 patients had multiple leiomyomas and we analyzed those multiple leiomyomas. Three of them had the same mutations. Five of them, each leiomyoma had a different mutation. Two of them did not have mutation. Four of them had both mutation-positive and mutation-negative leiomyomas. In conclusion, we confirmed the high frequency of the MED12 mutation in uterine leiomyomas of South Korean patients. We also identified various MED12 mutation status in patients with multiple leiomyomas. This suggests that in a given patient, different tumors may have arisen from different cell origins and therefore it is supposed that occurrence of multiple leiomyoma in a single patient may not be caused by intrauterine metastasis or dissemination.

ßTrCP-mediated ubiquitylation regulates protein stability of Mis18ß in a cell cycle-dependent manner.

Ubiquitin E3 ligases including SCF complex are key regulators of cell cycle. Here, we show that Mis18ß, a component of Mis18 complex governing CENP-A localization, is a new substrate of ßTrCP-containing SCF complex. ßTrCP interacted with Mis18ß exclusively during interphase but not during mitosis and mediated proteasomal degradation of Mis18ß leading to the inactivation of Mis18 complex during interphase. In addition, uncontrolled stabilization of Mis18ß caused cell death. Together, we propose that ßTrCP-mediated regulation of Mis18ß stability is a mechanism to restrict centromere function of Mis18 complex from late mitosis to early G1 phase.

Development of lumen-based perfusable 3D liver in vitro model using single-step bioprinting with composite bioinks.

Hepatic sinusoids are uniquely organized structures that help maintain a spectrum of hepatic functions. Although several in vitro liver models have been developed to replicate liver sinusoids, most of these platforms require complex, multi-step fabrication methods making it difficult to achieve truly three-dimensional (3D) channel geometries. In this study, a single-step bioprinting technique was demonstrated to simultaneously print a chip platform and develop a perfusable vascularized liver sinusoid in vitro model. The integrated system uses a co-axial-based bioprinting approach to develop a liver sinusoid-like model that consists of a sacrificial core compartment containing a perfusable pre-vascular structure and an alginate-collagen-based shell compartment containing hepatocytes. The lumen-based perfusable 3D liver sinusoid-on-a-chip (LSOC-P) demonstrated significantly better hepatocyte viability, proliferation, and liver-specific gene and protein expression compared to a 3D hepatocyte-based core/shell model with static media and the standard hepatocyte-based 2D sandwich culture system. A drug toxicity evaluation of hepatotoxins highlighted the comparatively higher sensitivity of the LSOC system with a close estimation of the therapeutic range of safe drug concentrations for humans. In conclusion, the current findings indicate that the combinatorial single-step co-axial bioprinting technique is a promising fabrication approach for the development of a perfusable LSOC platform for drug screening applications.

In planta expression screens of Phytophthora infestans RXLR effectors reveal diverse phenotypes, including activation of the Solanum bulbocastanum disease resistance protein Rpi-blb2.

The Irish potato famine pathogen Phytophthora infestans is predicted to secrete hundreds of effector proteins. To address the challenge of assigning biological functions to computationally predicted effector genes, we combined allele mining with high-throughput in planta expression. We developed a library of 62 infection-ready P. infestans RXLR effector clones, obtained using primer pairs corresponding to 32 genes and assigned activities to several of these genes. This approach revealed that 16 of the 62 examined effectors cause phenotypes when expressed inside plant cells. Besides the well-studied AVR3a effector, two additional effectors, PexRD8 and PexRD36(45-1), suppressed the hypersensitive cell death triggered by the elicitin INF1, another secreted protein of P. infestans. One effector, PexRD2, promoted cell death in Nicotiana benthamiana and other solanaceous plants. Finally, two families of effectors induced hypersensitive cell death specifically in the presence of the Solanum bulbocastanum late blight resistance genes Rpi-blb1 and Rpi-blb2, thereby exhibiting the activities expected for Avrblb1 and Avrblb2. The AVRblb2 family was then studied in more detail and found to be highly variable and under diversifying selection in P. infestans. Structure-function experiments indicated that a 34-amino acid region in the C-terminal half of AVRblb2 is sufficient for triggering Rpi-blb2 hypersensitivity and that a single positively selected AVRblb2 residue is critical for recognition by Rpi-blb2.

Stoichiometric Doping of Highly Coupled Cu2-xS Nanocrystal Assemblies.

The hole density of individual copper sulfide nanocrystals (Cu2-xS NCs) is determined from the stoichiometric mismatch (x) between copper and sulfide atoms. Consequently, the electronic properties of the material vary over a range of x. To exploit Cu2-xS NCs in devices, assemblies of NCs are typically required. Herein, we investigate the influence of x, referred to as the stoichiometric doping effect, on the structural, optical, electrical, and thermoelectric properties of electronically coupled Cu2-xS NC assemblies. The doping process is done by immersing the solid NC assemblies into a solution containing a Cu(I) complex for different durations (0-10 min). As Cu+ gradually occupied the copper-deficient sites of Cu2-xS NCs, x could be controlled from 0.9 to less than 0.1. Consequently, the near-infrared (NIR) absorbance of Cu2-xS NC assemblies changes systematically with x. With increasing x, electrical conductivity increased and the Seebeck coefficient decreased systematically, leading to the maximal thermoelectric power factor from a film of Cu2-xS NCs at an optimal doping condition yielding x = 0.1. The physical characteristics of the Cu2-xS NC assemblies investigated herein will provide guidelines for exploiting this emerging class of nanocrystal system based on doping.

Estimated Prevalence and Incidence of Uterine Leiomyoma, and Its Treatment Trend in South Korean Women for 12 years: A National Population-Based Study.

Background: Although uterine leiomyoma causes many problems, including infertility, there are few studies that have investigated the epidemiologic characteristics of uterine leiomyoma in South Korea. The aim of this study is to estimate the prevalence and incidence of uterine leiomyoma in South Korea and analyze the treatment trends. Materials and Methods: Women of reproductive age (15-54 years) were selected from the Korean National Health Insurance Service (NHIS) sample cohort dataset, which was collected from 2002 to 2013. Patients with uterine leiomyoma were identified by ICD-10 (International Codes of Disease, 10th Edition) and intervention codes. Prevalence and incidence were calculated from the NHIS cohort dataset and the treatment trends were analyzed for diagnosed patients. Results: The prevalence in overall age groups increased from 0.96% in 2002 to 2.43% in 2013, and the 1-year incidences of all age groups increased. The 26-30 age group showed the highest rate of 1-year incidence increase (2.14-folds, 0.33% in 2003 to 0.70% in 2013). The proportion of myomectomy increased from 22% in 2002 to 49% in 2013, whereas the proportion of hysterectomy decreased from 78% to 45%. Conclusions: The prevalence and incidence of uterine leiomyoma are increasing in South Korea as time progresses, and the rate of incidence increase is higher in younger reproductive women. Overall trends in uterine leiomyoma treatment are shifting to the methods of the saving uterus.

Clinical practice guidelines for radiofrequency ablation of benign thyroid nodules: a systematic review.

PURPOSE: Thermal ablation is a novel treatment alternative for benign thyroid nodules, and one of the most promising thermal ablation techniques is radiofrequency ablation (RFA). Considering the increasing use of thyroid RFA, some scientific societies have proposed clinical practice guidelines. We systemically reviewed and compared these guidelines for thyroid RFA to identify a standard treatment strategy that represents the positions of most societies. METHODS: We searched the MEDLINE and EMBASE databases for studies with human participants that were published in English between January 1, 2000 and August 2, 2019. Studies containing clinical practice guidelines for the RFA of benign thyroid nodules were included. We extracted data regarding indications, pre- and post-procedural evaluations, treatment techniques, and the need to obtain informed consent. RESULTS: Of the 83 studies found, four studies were included, and one study was added after searching the bibliographies of those articles. The five included studies were guidelines developed by the Korean Society of Thyroid Radiology, a group of experts from Italian scientific societies, the Italian Working Group on Minimally Invasive Treatments of the Thyroid, the United Kingdom's National Institute for Health and Clinical Excellence, and a group of four professional Austrian thyroid associations. Indications, pre- and post-procedural evaluations, and techniques were similar across studies; however, differences in each of these categories were found. CONCLUSION: While the reviewed guidelines are similar with regard to major categories, international guidelines for the RFA of benign thyroid nodules should be established in the future.

Polarization dependence suppression of optical fiber grating sensor in a π-shifted Sagnac loop interferometer.

In the sensing applications of optical fiber grating, it is necessary to reduce the transmission-type polarization dependence to isolate the sensing parameter. It is experimentally shown that the polarization-dependent spectrum of acousto-optic long-period fiber grating sensors can be suppressed in the transmission port of a π-shifted Sagnac loop interferometer. General expressions for the transmittance and reflectance are derived for transmission-type, reflection-type, and partially reflecting/transmitting-type polarization-dependent optical devices. The compensation of polarization dependence through the counter propagation in the Sagnac loop interferometer is quantitatively measured for a commercial in-line polarizer and an acousto-optic long-period fiber grating sensor.

Antiproliferative Effect of 4-Methylumbelliferone in Epithelial Ovarian Cancer Cells Is Mediated by Disruption of Intracellular Homeostasis and Regulation of PI3K/AKT and MAPK Signaling.

Ovarian cancer has a high mortality rate and high resistance to chemotherapy. Thus, many studies are currently assessing the ability of natural products to induce ovarian cancer cell death. A coumarin derivative, 4-methylumbelliferone (4-MU), has been reported to have anti-cancer effects on various cancers, but its effects on ovarian cancer are not fully understood. In this study, we identified the intracellular mechanism underlying the effects of 4-MU on epithelial ovarian cancer cells. Decreased ovarian cancer cell proliferation and an accumulation of cells in the G2/M phase were observed following 4-MU treatment. Moreover, 4-MU interfered with calcium homeostasis; induced endoplasmic reticulum stress in both cell lines; inhibited AKT and S6 phosphorylation; and increased ERK1/2, P38, and JNK phosphorylation. Furthermore, 4-MU and pharmacological inhibitors showed synergic effects in suppressing cell proliferation. Collectively, our current data indicate that antitumor effects of 4-MU could be appropriate for use as a therapeutic agent against epithelial ovarian cancer cells.

High-resolution patterning of colloidal quantum dots via non-destructive, light-driven ligand crosslinking.

Establishing multi-colour patterning technology for colloidal quantum dots is critical for realising high-resolution displays based on the material. Here, we report a solution-based processing method to form patterns of quantum dots using a light-driven ligand crosslinker, ethane-1,2-diyl bis(4-azido-2,3,5,6-tetrafluorobenzoate). The crosslinker with two azide end groups can interlock the ligands of neighbouring quantum dots upon exposure to UV, yielding chemically robust quantum dot films. Exploiting the light-driven crosslinking process, different colour CdSe-based core-shell quantum dots can be photo-patterned; quantum dot patterns of red, green and blue primary colours with a sub-pixel size of 4 µm × 16 µm, corresponding to a resolution of >1400 pixels per inch, are demonstrated. The process is non-destructive, such that photoluminescence and electroluminescence characteristics of quantum dot films are preserved after crosslinking. We demonstrate that red crosslinked quantum dot light-emitting diodes exhibiting an external quantum efficiency as high as 14.6% can be obtained.

Effects of fermentation on SDS-PAGE patterns, total peptide, isoflavone contents and antioxidant activity of freeze-thawed tofu fermented with Bacillus subtilis.

To develop a new healthy functional tofu product, the pH, total cell number, SDS-PAGE patterns, contents of reducing sugar, peptide, isoflavones, antioxidant activity and digestibility of freeze-thawed tofu, fermented with Bacillus subtilis for various time periods, were investigated. In SDS-PAGE patterns, the band intensities of 7S and 11S globulins were slightly decreased and new protein bands of lower molecular weight appeared by fermentation for 12 h. After 18 h, the bands of 7S globulin and the acidic subunit of 11S globulin had almost entirely disappeared and the basic subunit band of 11S globulin was fainter. An 18 h of fermentation was thought to be the most desirable, because the contents of reducing sugar, total peptide, and isoflavone aglycones (daidzein and genistein), as well as antioxidant activities and digestibility of freeze-thawed tofu were more increased by the fermentation for 18 h.

ULK1 O-GlcNAcylation Is Crucial for Activating VPS34 via ATG14L during Autophagy Initiation.

Unc-51-like-kinase 1 (ULK1) is a target of both the mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK), whose role is to facilitate the initiation of autophagy in response to starvation. Upon glucose starvation, dissociation of mTOR from ULK1 and phosphorylation by AMPK leads to the activation of ULK1 activity. Here, we provide evidence that ULK1 is the attachment of O-linked N-acetylglucosamine (O-GlcNAcylated) on the threonine 754 site by O-linked N-acetylglucosamine transferase (OGT) upon glucose starvation. ULK1 O-GlcNAcylation occurs after dephosphorylation of adjacent mTOR-dependent phosphorylation on the serine 757 site by protein phosphatase 1 (PP1) and phosphorylation by AMPK. ULK1 O-GlcNAcylation is crucial for binding and phosphorylation of ATG14L, allowing the activation of lipid kinase VPS34 and leading to the production of phosphatidylinositol-(3)-phosphate (PI(3)P), which is required for phagophore formation and initiation of autophagy. Our findings provide insights into the crosstalk between dephosphorylation and O-GlcNAcylation during autophagy and specify a molecular framework for potential therapeutic intervention in autophagy-related diseases.