O-GlcNAcylation stabilizes the autophagy-initiating kinase ULK1 by inhibiting chaperone-mediated autophagy upon HPV infection.

Human papillomaviruses (HPVs) cause a subset of head and neck squamous cell carcinomas (HNSCCs). Previously, we demonstrated that HPV16 oncogene E6 or E6/E7 transduction increases the abundance of O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT), but OGT substrates affected by this increase are unclear. Here, we focus on the effects of O-GlcNAcylation on HPV-positive HNSCCs. We found that upon HPV infection, Unc-51-like kinase 1 (ULK1), an autophagy-initiating kinase, is hyper-O-GlcNAcylated, stabilized, and linked with autophagy elevation. Through mass spectrometry, we identified that ULK1 is O-GlcNAcylated at Ser409, which is distinct from the previously reported Thr635/Thr754 sites. It has been demonstrated that PKCα mediates phosphorylation of ULK1 at Ser423, which attenuates its stability by shunting ULK1 to the chaperone-mediated autophagy (CMA) pathway. Using biochemical assays, we demonstrate that ULK1 Ser409Ser410 O-GlcNAcylation antagonizes its phosphorylation at Ser423. Moreover, mutations of Ser409A and its neighboring site Ser410A (2A) render ULK1 less stable by promoting interaction with the CMA chaperone HSC70 (heat shock cognate 70 kDa protein). Furthermore, ULK1-2A mutants attenuate the association of ULK1 with STX17, which is vital for the fusion between autophagosomes and lysosomes. Analysis of The Cancer Genome Atlas (TCGA) database reveals that ULK1 is upregulated in HPV-positive HNSCCs, and its level positively correlates with HNSCC patient survival. Overall, our work demonstrates that O-GlcNAcylation of ULK1 is altered in response to environmental changes. O-GlcNAcylation of ULK1 at Ser409 and perhaps Ser410 stabilizes ULK1, which might underlie the molecular mechanism of HPV-positive HNSCC patient survival.

MCAD activation by empagliflozin promotes fatty acid oxidation and reduces lipid deposition in NASH.

Medium-chain acyl-CoA dehydrogenase (MCAD) is one of the significant enzymes involved in the ß-oxidation of mitochondrial fatty acids. MCAD deficiency affects the ß-oxidation of fatty acid and leads to lipid deposition in multiple organs, but little is known about its importance in nonalcoholic steatohepatitis (NASH). Empagliflozin is revealed to effectively improve NASH by increasing research, whereas the specific mechanism still has to be explored. Human liver tissues of patients with or without NASH were obtained for proteomic analysis to screen proteins of interest. db/db mice were given empagliflozin by gavage for 8 weeks. The expression of MCAD and signaling molecules involved in hepatic lipid metabolism was evaluated in human liver, mice and HL7702 cells. We found that the MCAD levels in the liver were significantly reduced in NASH patients compared to patients without NASH. Protein-protein interaction network analysis showed that MCAD was highly correlated with forkhead box A2 (FOXA2) and protein kinase AMP-activated catalytic subunit alpha (PRKAA). AMPK/FOXA2/MCAD signaling pathway was detected to be inhibited in the liver of NASH patients. Decreased expression of MCAD was also observed in the livers of db/db mice and hepatocyte treated with palmitic acid and glucose. Of note, empagliflozin could upregulate MCAD expression by activating AMPK/FOXA2 signaling pathway, reduce lipid deposition and improve NASH in vivo and in vitro. This research demonstrated that MCAD is a key player of hepatic lipid deposition and its targeting partially corrects NASH. MCAD thus may be a potential therapeutic target for the treatment of NASH.

High-Sensitivity C-Reactive Protein and Ischemic Stroke in Patients with Nonalcoholic Fatty Liver Disease: A Prospective Study.

Background and Aims: Inflammation is involved in the pathophysiology of ischemic stroke. The aim of this prospective study was to evaluate the association of hs-CRP with incident ischemic stroke in patients with nonalcoholic fatty liver disease (NAFLD). Methods: A sample of 318 participants without previous strokes was included in this study. Hs-CRP levels and other potential confounding factors were measured at baseline. NAFLD was performed by abdominal ultrasound after excluding secondary causes for fat accumulation. According to baseline hs-CRP concentrations, participants were categorized into 3 groups: level 1 (<1.0 mg/L), level 2 (1.0 to <3.0 mg/L), and level 3 (≥3.0 mg/L). The outcome of interest was the first occurrence of an ischemic stroke. Cox proportional hazards models were used to analyze hazard ratios (HRs) and 95% confidence intervals (CIs) of incident ischemic stroke, after adjusting for potential confounders. Results: The mean age of 318 participants with NAFLD was 71.1 ± 6.7 years, and 55.3% of them were male. Among 318 individuals with NAFLD, 115 (36.2%) of them had an hs-CRP value <1 mg/L (level 1), 105 (33.0%) had an hs-CRP value between 1 and 3 mg/L (level 2), and 98 (30.8%) belonged to level 3 (hs-CRP ≥3 mg/L). Over a median of 5.60 years of follow-up, 47 incident ischemic stroke events were documented in 318 patients with NAFLD. After full adjustment for confounding factors, compared with participants in the level 1 group (hs-CRP<1.0 mg/L), the HRs of those in the level 2 group (1.0 to <3.0 mg/L) and the level 3 group (≥3.0 mg/L) were 1.77 (95% CI: 0.94-2.98) and 2.45 (95% CI: 1.37-5.77) for developing ischemic stroke, respectively. Conclusions: Elevated hs-CRP levels were associated with an increased risk of ischemic stroke among patients with NAFLD.

Sarcopenia as an independent predictor for venous thromboembolism events in bladder cancer patients undergoing radical cystectomy.

BACKGROUND: Sarcopenia has been proved to be related to the prognosis of patients with bladder cancer (BC) after radical cystectomy (RC). The relationship between sarcopenia and the occurrence of venous thromboembolism (VTE) after RC is unclear. METHODS: We collected data of 252 BC patients treated with RC at our institution. Data was obtained from the electronic medical record database. Sarcopenia was defined by the third lumbar vertebra skeletal muscle index (SMI) which was measured using preoperative computed tomography. The primary outcome was the incidence of VTE within 30 days after the surgery in sarcopenia and non-sarcopenia groups. Outcomes between the two cohorts were compared using univariate analysis. Multivariate logistic regression was used to control for differences between cohorts. RESULTS: Two hundred fifty-two patients were enrolled, of which 85 (33.7%) patients were in sarcopenia group, while 167 (66.3%) patients were not in sarcopenia group. The incidence of total VTE in sarcopenia group was higher than that in the extended group (10.6% vs. 1.8%, p = 0.005). Sarcopenia did not cause an increase in other postoperation 30 days complications (all p > 0.05). Multivariate analysis confirmed sarcopenia was independently associated with increased odds of VTE (OR = 4.18, 95% CI [1.01-17.27]; p = 0.048). Subgroup analysis showed that patients with VTE tended to be older (76.5 vs 66.0, p = 0.025) and have higher proportion of diabetes (58.3% vs 14.2%, p < 0.001) as well as lower level of serum albumin (35.0 g/L vs 40.4 g/L, p = 0.023) compared with those without VTE. CONCLUSIONS: Sarcopenia was an independent predictor for VTE with patients undergoing RC for BC.

SDF-1 inhibits the dedifferentiation of islet ß cells in hyperglycaemia by up-regulating FoxO1 via binding to CXCR4.

Islet ß cell dedifferentiation is one of the most important mechanisms in the occurrence and development of diabetes. We studied the possible effects of chemokine stromal cell-derived factor-1 (SDF-1) in the dedifferentiation of islet ß cells. It was noted that the number of dedifferentiated islet ß cells and the expression of SDF-1 in pancreatic tissues significantly increased with diabetes. In islet ß cell experiments, inhibition of SDF-1 expression resulted in an increase in the number of dedifferentiated cells, while overexpression of SDF-1 resulted in a decrease. This seemed to be contradicted by the effect of diabetes on the expression of SDF-1 in pancreatic tissue, but it was concluded that this may be related to the loss of SDF-1 activity. SDF-1 binds to CXCR4 to form a complex, which activates and phosphorylates AKT, subsequently increases the expression of forkhead box O1 (FOXO1), and inhibits the dedifferentiation of islet ß cells. This suggests that SDF-1 may be a novel target in the treatment of diabetes.

Visceral fat area to appendicular muscle mass ratio as a predictor for nonalcoholic fatty liver disease independent of obesity.

OBJECTIVE: Obesity and sarcopenia are known to be closely related to nonalcoholic fatty liver disease (NAFLD). We attempted to explore the combined influence of fat and muscle tissue on NAFLD by using visceral fat area to appendicular muscle mass ratio (VAR) as a novel parameter. MATERIAL AND METHODS: In this cross-sectional study, a total of 3255 adults (1399 men and 1856 women) coming for a health examination were enrolled. NAFLD was diagnosed using ultrasound and VAR was measured by bioelectrical impedance analyzer. RESULTS: The prevalence of NAFLD was 46.5% in men and 26.6% in women. VAR differed significantly between subjects with and without NAFLD (4.27 vs. 3.26 in men, 7.89 vs. 5.01 in women, respectively, p < .001). Logistic regression analysis determined VAR as a risk factor for NAFLD, and the multivariable-adjusted odds ratios in the highest VAR quartile was 9.57 (95%CI: 5.98-15.30) for men and 12.37 (95%CI: 6.37-24.05) for women. From the receiver operating characteristic analysis, the area under the curve was 0.767 and 0.834, with the suitable cut-off VAR value of 3.469 and 6.357 for men and women, respectively. To control the influence of obesity, all subjects were stratified according to their BMI. For each BMI group, individuals with VAR above the cut-off value had significant higher prevalence and risk of NAFLD, with odds ratios ranging from 1.76 to 4.75. CONCLUSIONS: Increased VAR is strongly associated with higher risk of NAFLD in both sexes independent of obesity and can serve as a screening reference for NAFLD.

The visceral fat area to leg muscle mass ratio is significantly associated with the risk of hyperuricemia among women: a cross-sectional study.

BACKGROUND: A significant positive association was found in previous studies among obesity, visceral fat accumulation, and hyperuricemia. The purpose of this study was to explore the association between the ratio of visceral fat area to leg muscle mass (VFA-to-LMM) and hyperuricemia, and verify the role of gender differences in the association. METHODS: A total of 3393 (43.3% are men) participants from Tianjin Union Medical Center-Health Management Center were recruited for this cross-sectional study. The VFA-to-LMM ratio was used as the independent variable. Hyperuricemia, a serum uric acid level ≥ 416 µmol/L in men and in menopausal women and ≥ 357 µmol/L in premenopausal women, was used as the dependent variable. Multiple logistic regression analysis was used to estimate the odds ratio and the 95% confidence interval between the VFA-to-LMM ratio and hyperuricemia. RESULTS: The overall prevalence of hyperuricemia was 14.8% (8.9% in women, and 22.5% in men). After adjustment by age, smoking status (for males), menopause status (for females), drinking status, exercise frequency, blood pressure, alanine aminotransferase, fasting plasma glucose, triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, creatinine, and history of diseases, a strong positive association was found between the VFA-to-LMM ratio and hyperuricemia in both men (4th vs. 1st quartile 1.60, 95%CI: 1.03-2.49) and women (4th vs. 1st quartile 5.22, 95%CI: 2.44-12.56). After additional adjustment by BMI, there was still a significant positive association in women (4th vs. 1st quartile 2.57, 95%CI: 1.06-6.77). The results of subgroup analysis showed that pre-menopausal women (4th vs. 1st quartile OR: 3.61) have a higher risk of hyperuricemia than postmenopausal women (4th vs. 1st quartile OR: 1.94) with the increase of the VFA-to-LMM ratio. Besides, the interaction analysis results showed the highest risk of hyperuricemia when VFA and LMM were both in the highest quantile (OR: 11.50; 95% CI: 4.86-31.98). CONCLUSION: The VFA-to-LMM ratio was positively associated with the risk of hyperuricemia in women after adjustment by confounders. Pre-menopausal women have a higher risk of hyperuricemia than postmenopausal women with the increase of the VFA-to-LMM ratio. In addition, the highest risk of hyperuricemia was demonstrated when both VFA and LMM were at the highest quartile.

O-GlcNAcylation of myosin phosphatase targeting subunit 1 (MYPT1) dictates timely disjunction of centrosomes.

The role of O-linked N-acetylglucosamine (O-GlcNAc) modification in the cell cycle has been enigmatic. Previously, both O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) disruptions have been shown to derail the mitotic centrosome numbers, suggesting that mitotic O-GlcNAc oscillation needs to be in concert with mitotic progression to account for centrosome integrity. Here, using both chemical approaches and biological assays with HeLa cells, we attempted to address the underlying molecular mechanism and observed that incubation of the cells with the OGA inhibitor Thiamet-G strikingly elevates centrosomal distances, suggestive of premature centrosome disjunction. These aberrations could be overcome by inhibiting Polo-like kinase 1 (PLK1), a mitotic master kinase. PLK1 inactivation is modulated by the myosin phosphatase targeting subunit 1 (MYPT1)-protein phosphatase 1cß (PP1cß) complex. Interestingly, MYPT1 has been shown to be abundantly O-GlcNAcylated, and the modified residues have been detected in a recent O-GlcNAc-profiling screen utilizing chemoenzymatic labeling and bioorthogonal conjugation. We demonstrate here that MYPT1 is O-GlcNAcylated at Thr-577, Ser-585, Ser-589, and Ser-601, which antagonizes CDK1-dependent phosphorylation at Ser-473 and attenuates the association between MYPT1 and PLK1, thereby promoting PLK1 activity. We conclude that under high O-GlcNAc levels, PLK1 is untimely activated, conducive to inopportune centrosome separation and disruption of the cell cycle. We propose that too much O-GlcNAc is equally deleterious as too little O-GlcNAc, and a fine balance between the OGT/OGA duo is indispensable for successful mitotic divisions.

Chk2-dependent phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) regulates centrosome maturation.

Checkpoint kinase 2 (Chk2) is a pivotal effector kinase in the DNA damage response, with an emerging role in mitotic chromosome segregation. In this study, we show that Chk2 interacts with myosin phosphatase targeting subunit 1 (MYPT1), the targeting subunit of protein phosphatase 1cß (PP1cß). Previous studies have shown that MYPT1 is phosphorylated by CDK1 at S473 during mitosis, and subsequently docks to the polo-binding domain of PLK1 and dephosphorylates PLK1. Herein we present data that Chk2 phosphorylates MYPT1 at S507 in vitro and in vivo, which antagonizes pS473. Chk2 inhibition results in failure of γ-tubulin recruitment to the centrosomes, phenocopying Plk1 inhibition defects. These aberrancies were also observed in the MYPT1-S507A stable transfectants, suggesting that Chk2 exerts its effect on centrosomes via MYPT1. Collectively, we have identified a Chk2-MYPT1-PLK1 axis in regulating centrosome maturation. Abbreviations: Chk2: checkpoint kinase 2; MYPT1: myosin phosphatase targeting subunit 1; PP1cß: protein phosphatase 1c ß; Noc: nocodazole; IP: immunoprecipitation; IB: immunoblotting; LC-MS/MS: liquid chromatography-tandem mass spectrometry; Chk2: checkpoint kinase 2; KD: kinase domain; WT: wild type; Ub: ubiquitin; DAPI: 4',6-diamidino-2-phenylindole; IF: Immunofluorescence; IR: ionizing radiation; siCHK2: siRNA targeting CHK2.

Efficacy and safety of noninvasive ventilation in patients after cardiothoracic surgery: A PRISMA-compliant systematic review and meta-analysis.

BACKGROUND: Noninvasive ventilation (NIV) is a promising therapeutic strategy after cardiothoracic surgery. This study aimed to meta-analyze the efficacy and safety of NIV as compared to conventional management after cardiothoracic surgery. METHODS: PubMed, EMBASE, and Cochrane Library databases were searched for randomized controlled trials (RCTs) comparing NIV with conventional management after cardiothoracic surgery. Relative risk (RR), standard mean difference (SMD), and 95% confidence intervals (CIs) were used to measure the efficacy and safety of NIV using random-effects model. Heterogeneity was evaluated using the Q statistic. RESULTS: This study included 14 RCTs (1740 patients) for the evaluation of efficacy and safety of NIV as compared to conventional management after cardiothoracic surgery. Overall, NIV had minimal effect on the risk of mortality (RR: 0.64; 95% CI: 0.36-1.14; P = 0.127), endotracheal intubation (RR: 0.52; 95% CI: 0.24-1.11; P = 0.090), respiratory (RR: 0.70; 95% CI: 0.47-1.30; P = 0.340), cardiovascular (RR: 0.81; 95% CI: 0.54-1.22; P = 0.306), renal (RR: 0.70; 95% CI: 0.26-1.92; P = 0.491), and other complications (RR: 0.72; 95% CI: 0.38-1.36; P = 0.305), respiratory rate (SMD: -0.10; 95% CI: -1.21-1.01; P = 0.862), heart rate (SMD: -0.27; 95% CI: -0.76-0.22; P = 0.288), PaO2/FiO2 ratio (SMD: 0.34; 95% CI: -0.17-0.85; P = 0.194), PaCO2 (SMD: 0.83; 95% CI: -0.12-1.77; P = 0.087), systolic pressure (SMD: -0.04; 95% CI: -0.25-0.17; P = 0.700), pH (SMD: -0.01; 95% CI: -0.44-0.43; P = 0.974), length of ICU stay (SMD: -0.19; 95% CI: -0.47-0.08; P = 0.171), and hospital stay (SMD: -0.31; 95% CI: -1.00-0.38; P = 0.373). Sensitivity analysis showed that NIV was associated with higher levels of PaO2/FiO2 ratio (SMD: 0.52; 95% CI: 0.00-1.05; P = 0.048) and lower risk of endotracheal intubation (RR: 0.38; 95% CI: 0.22-0.66; P = 0.001). CONCLUSION: As compared to conventional management, the use of NIV after cardiothoracic surgery improved patient's oxygenation and decreased the need for endotracheal intubation, without significant complications.

Activation of G-Protein-Coupled Estrogen Receptor Inhibits the Migration of Human Nonsmall Cell Lung Cancer Cells via IKK-ß/NF-κB Signals.

Estrogen signals have been suggested to modulate the progression and metastasis of nonsmall cell lung cancer (NSCLC), which is one of the leading causes of cancer deaths worldwide. While there are limited data concerning the roles and effects of G-protein-coupled estrogen receptor (GPER) on the progression of NSCLC, our present study reveals that the expression of GPER in NSCLC cells is obviously greater than that in lung fibroblast cell line MRC-5. Activation of GPER via its specific agonist G-1 decreases the in vitro motility of A549 and H358 cells and the expression of matrix metalloproteinase 2 (MMP-2) and MMP-9. Further, G-1 treatment can rapidly decrease the phosphorylation, nuclear translocation, and promoter activities of NF-κB in NSCLC cells. BAY 11-7082, the inhibitor of NF-κB, also inhibits the expression of MMP-2/9, while overexpression of p65 significantly attenuates G-1-induced downregulation of MMP-2/9. It suggests that inhibition of NF-κB mediates G-1-induced MMP-2/9 downregulation. G-1 treatment significantly down regulates the phosphorylation of IκB kinase ß (IKK-ß) and IκBα, while not IKK-α, in both 549 and H358 cells. ACHP, the specific inhibitor of IKK-ß, can reinforce G-1-induced MMP-2/9 downregulation and invasion suppression of A549 cells. Collectively, our results suggest that activation of GPER can inhibit the migration of human NSCLC cells via suppression of IKK-ß/NF-κB signals. These findings will help to better understand the roles and mechanisms of GPER as a potential therapy target for NSCLC patients.

[Composition and distribution of TCBS bacteria groups from sediments of Jiulong River estuary].

OBJECTIVE: To investigate potential pathogens in waters of Xiamen from Jiulong River, and to provide useful information for the prevention and control of potential pathogen infections. METHODS: All samples were spread on Thiosulfate Citrate Bile Salts Sucrose (TCBS) agar plates, and then incubated at 26 +/- 1 degrees C for 24 +/- 2 h. In total 158 TCBS strains were isolated from TCBS agar plates and pure-cultivated on 2216E agar plates. All strains were identified using the 16S rRNA gene- Restriction fragment length polymorphism (RFLP), 16S rRNA sequence analysis, GenBank database Basic Local Alignment Search Tool (BLAST) and phylogenetic analysis. RESULTS: The results show that 158 TCBS strains from the sediments of Jiulong River estuary were classfied as 7 genus, which were Pseudomonas (28%), Aeromonas (24%), Pseudoalteromonas (19%), Shewanella (13%), Bacillus (11%), Vibrio (4%) and Psychrobacter (1%). The composition and distribution of TCBS bacteria groups varied with stations. Non-halophilic or haloduric bacteria groups were dominant in the upper area of Jiulong River estuary, and halophilic and haloduric bacteria were dominant in the lower area, which characterized a typical estuary feature. The salinity played a key role in the distribution of TCBS groups. Vibrios did not constitute a significant proportion (6% - 19%) of the total TCBS strains at different stations, and most of the them distributed at the lower region. CONCLUSION: There were a lot of potential pathogens in Jiulong River estuary. Aeromonas, a typical genus of halotolerant bacteria, was the potentially terrigenous bacteria contamination to the waters of Xiamen. Most Vibrio specieses were marine aborigines, which was not directly contaminated from the runoff of Jiulong River.