GABARAPs and LC3s have opposite roles in regulating ULK1 for autophagy induction.

ULK1 (unc-51 like autophagy activating kinase 1) is the key mediator of MTORC1 signaling to macroautophagy/autophagy. ULK1 functions as a protein complex by interacting with ATG13, RB1CC1/FIP200, and ATG101. How the ULK1 complex is regulated to trigger autophagy induction remains unclear. In this study, we have determined roles of Atg8-family proteins (ATG8s) in regulating ULK1 activity and autophagy. Using human cells depleted of each subfamily of ATG8, we found that the GABARAP subfamily positively regulates ULK1 activity and phagophore and autophagosome formation in response to starvation. In contrast, the LC3 subfamily negatively regulates ULK1 activity and phagophore formation. By reconstituting ATG8-depleted cells with individual ATG8 members, we identified GABARAP and GABARAPL1 as positive and LC3B and LC3C as negative regulators of ULK1 activity. To address the role of ATG8 binding to ULK1, we mutated the LIR of endogenous ULK1 to disrupt the ATG8-ULK1 interaction by genome editing. The mutation drastically reduced the activity of ULK1, autophagic degradation of SQSTM1, and phagophore formation in response to starvation. The mutation also suppressed the formation and turnover of autophagosomes in response to starvation. Similar to the mutation of the ULK1 LIR, disruption of the ATG13-ATG8 interaction suppressed ULK1 activity and autophagosome formation. In contrast, RB1CC1 did not show any specific binding to ATG8s, and mutation of its LIR did not affect ULK1 activity. Together, this study demonstrates differential binding and opposite regulation of the ULK1 complex by GABARAPs and LC3s, and an important role of the ULK1- and ATG13-ATG8 interactions in autophagy induction.Abbreviations: ATG5: autophagy related 5; ATG7: autophagy related 7; ATG8: autophagy related 8; ATG13: autophagy related 13; ATG14: autophagy related 14; ATG16L1: autophagy related 16 like 1; ATG101: autophagy related 101; BAFA1: bafilomycin A1; BECN1: beclin 1; Cas9: CRISPR associated protein 9; CRISPR: clustered regularly interspaced short palindromic repeats; EBSS: earle's balanced salt solution; DAPI: 4'-6-diamidino-2-phenylindole; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor-associated protein like 1; GABARAPL2: GABA type A receptor-associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescence protein; gRNA: guide RNA; KI: kinase inactive mutant; KO: knockout; LC3A: microtubule associated protein 1 light chain 3 alpha; LC3B: microtubule associated protein 1 light chain 3 beta; LC3C: microtubule associated protein 1 light chain 3 gamma; LIR: LC3-interacting region; MTORC1: mechanistic target of rapamycin kinase complex 1; PBS: phosphate buffered saline; PCR: polymerase chain reaction; PE: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; qPCR: quantitative PCR; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RPS6KB1: ribosomal protein S6 kinase B1; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TALEN: transcription activator-like effector nuclease; TUBA: tubulin alpha; ULK1: unc-51 like autophagy activating kinase 1; WB: western blotting; WIPI2: WD repeat domain phosphoinositide interacting 2; WT: wild type.

Further Defining the 2012 Multi-Society Task Force Guidelines for Surveillance of High-risk Adenomas: Is a 3-Year Interval Needed for All Patients?

GOALS: We set out to determine whether variation from this 3-year follow-up interval was associated with the finding of subsequent high-risk adenoma (HRA). BACKGROUND: HRAs include the following: (1) an adenoma measuring ≥10 mm, (2) ≥3 adenomas found during a single procedure, and (3) an adenoma with high-grade dysplasia or villous architecture. The current Multi-Society Task Force guideline for timing of surveillance colonoscopy after removal of a HRA is 3 years. STUDY: In 2016, we analyzed 495 patients who had a HRA removed during a 2008 colonoscopy. We compared the frequency of finding another HRA at follow-up intervals. We used the current guidelines as our referent group and performed logistical regression to identify whether any patient characteristics, procedural factors, or type of HRA predicted the development of HRAs on follow-up colonoscopy. RESULTS: Individuals who followed-up at a median of 4.5 years did not have more HRA on follow-up compared with those who followed-up at 3 years (25.2% vs. 21.0%, P=0.062). These groups had similar baseline characteristics. Older individuals, male gender, having a history of polyps, and piecemeal resection of an HRA predicted future HRAs. The removal of ≥3 adenomas in 2008 as well as a combination of multiple, large, and advanced polyps showed a higher risk of future HRAs. CONCLUSIONS: The 2012 Multi-Society Task Force recommendation of 3-year follow-up after removal of HRAs may not apply to all patients. We showed that a combination of patient demographics, procedural factors, and pathology best determines the surveillance colonoscopy interval.

ULK1 phosphorylates Ser30 of BECN1 in association with ATG14 to stimulate autophagy induction.

ULK1 (unc51-like autophagy activating kinase 1) is a serine/threonine kinase that plays a key role in regulating macroautophagy/autophagy induction in response to amino acid starvation. Despite the recent progress in understanding ULK1 functions, the molecular mechanism by which ULK1 regulates the induction of autophagy remains elusive. In this study, we determined that ULK1 phosphorylates Ser30 of BECN1 (Beclin 1) in association with ATG14 (autophagy-related 14) but not with UVRAG (UV radiation resistance associated). The Ser30 phosphorylation was induced by deprivation of amino acids or treatments with Torin 1 or rapamycin, the conditions that inhibit MTORC1 (mechanistic target of rapamycin complex 1), and requires ATG13 and RB1CC1 (RB1 inducible coiled-coil 1), proteins that interact with ULK1. Hypoxia or glutamine deprivation, which inhibit MTORC1, was also able to increase the phosphorylation in a manner dependent upon ULK1 and ULK2. Blocking the BECN1 phosphorylation by replacing Ser30 with alanine suppressed the amino acid starvation-induced activation of the ATG14-containing PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) kinase, and reduced autophagy flux and the formation of phagophores and autophagosomes. The Ser30-to-Ala mutation did not affect the ULK1-mediated phosphorylations of BECN1 Ser15 or ATG14 Ser29, indicating that the BECN1 Ser30 phosphorylation might regulate autophagy independently of those 2 sites. Taken together, these results demonstrate that BECN1 Ser30 is a ULK1 target site whose phosphorylation activates the ATG14-containing PIK3C3 complex and stimulates autophagosome formation in response to amino acid starvation, hypoxia, and MTORC1 inhibition.

A National Survey of the Prevalence and Impact of Cytomegalovirus Infection Among Hospitalized Patients With Ulcerative Colitis.

GOALS: To estimate the effect of cytomegalovirus (CMV) in patients with ulcerative colitis (UC), and compare these outcomes to patients with CMV without UC. BACKGROUND: The impact of CMV infection in UC is not well understood. STUDY: We analyzed records from the Nationwide Inpatient Sample (NIS) of patients with UC and CMV between 2006 and 2012. Differences in outcomes were determined between patients with UC and CMV and those with UC without CMV. Secondary analysis compared outcomes of patients with UC and CMV to patients with CMV alone. RESULTS: Patients with UC and CMV (n=145) had longer length of stay (16.31 vs. 5.52 d, P<0.0001), higher total charges ($111,835.50 vs. $39.895, P=0.001), and were less likely to be discharged home without services (50.0% vs. 81.83%, P<0.0001) compared with patients with UC without CMV (n=32,290). On regression analysis, CMV was significantly associated with higher total charges (P<0.01) and longer length of stay (P<0.01), but not for increased need for colorectal surgery. When comparing patients with UC and CMV to patients with CMV alone (n=14,960), patients with CMV alone had a higher Charlson Comorbidity Index and a trend toward higher in-hospital mortality. CONCLUSIONS: CMV infection in hospitalized patients with UC is associated with a longer length of stay, increased total charges, and fewer routine discharges, but not increased surgery or mortality. Patients with CMV alone had the worst outcomes of all groups suggesting that CMV in UC patients may not have the same negative impact as in other diseases.

Comparison of Vascular Plugs and Pushable Coils for Variceal Embolization After TIPS.

OBJECTIVE: Transjugular intrahepatic portosystemic shunt (TIPS) with variceal embolization is routinely performed to treat variceal bleeding. Embolization using vascular plugs is reported, but outcomes are not known. Outcomes and material costs of embolization using vascular plugs and coils are compared. MATERIALS AND METHODS: A single center's medical records of TIPS procedures (May 2003-December 2014) with variceal embolization were reviewed. Twenty patients with vascular plug embolization (age [± SD], 50 ± 10 years; seven men and 13 women; median Model for End-Stage Liver Disease [MELD], 20; interquartile range [IQR], 14-23) were compared with an age-, sex-, and MELD-matched cohort who underwent coil embolization (age, 50 ± 9 years; seven men and 13 women; median MELD, 17; IQR, 15-19; p = 0.52). Procedure details, primary outcome (rebleeding), secondary outcome (mortality), and costs were compared. RESULTS: Vascular plug use was associated with a lower fluoroscopy time (49.05 minutes [IQR, 36-62] vs 68 minutes [IQR, 49-76]; p = 0.006) and total procedure time (255 minutes [IQR, 205-290] for vascular plugs vs 275 minutes [IQR, 230-330]; p = 0.05). Total volume of contrast agent used was similar (180 mL [IQR, 155-234] for vascular plugs vs 210 mL [IQR, 185-261]; p = 0.14). In patients with at least a 30-day follow-up, rebleeding rates (2/17 [12%] for vascular plugs vs 4/15 [27%]; p = 0.40) and mortality (2/17 [12%] for vascular plugs vs 4/15 [27%]; p = 0.66) were similar. Per procedure, vascular plugs cost significantly more than coils ($1292 ± $676 vs $228 ± $292, p < 0.0001). CONCLUSION: The use of vascular plugs or coils has similar outcomes for variceal embolization after TIPS. The advantages of vascular plug use (i.e., reduced fluoroscopy or procedure time) may be offset by increased material cost, a trade-off that merits further study given current cost concerns in health care.

The Effect of Right Colon Retroflexion on Adenoma Detection: A Systematic Review and Meta-analysis.

BACKGROUND: Although colonoscopy with polypectomy can prevent up to 80% of colorectal cancers, a significant adenoma miss rate still exists, particularly in the right colon. Previous studies addressing right colon retroflexion have revealed discordant evidence regarding the benefit of this maneuver on adenoma detection with concomitant concerns about safety and rates of maneuver success. In this meta-analysis, we sought to determine the effect of right colon retroflexion on improving adenoma detection compared with conventional colonoscopy without retroflexion, as well as determine the rates of retroflexion maneuver success and adverse events. METHODS: Multiple databases including MEDLINE, Embase, and Web of Science were searched for studies on right colon retroflexion and its impact on adenoma detection compared with conventional colonoscopy. Pooled analyses of adenoma detection and retroflexion success were based on mixed-effects and random-effects models with heterogeneity analyses. RESULTS: Eight studies met the inclusion criteria (N=3660). The primary analysis comparing colonoscopy with right-sided retroflexion versus conventional colonoscopy to determine the per-adenoma miss rate in the right colon was 16.9% (95% confidence interval, 12.5%-22.5%). The overall rate of successful retroflexion was 91.9% (95% confidence interval, 86%-95%) and rate of adverse events was 0.03%. CONCLUSIONS: Colonoscopy with right-sided retroflexion significantly increases the detection of adenomas in the right colon compared with conventional colonoscopy with a high rate of maneuver success and small risk of adverse events. Thus, reexamination of the right colon in retroflexed view should be strongly considered in future standard of care colonoscopy guidelines for quality improvement in colon cancer prevention.

An Analysis of Factors Predicting Successful Transition From Pancreatology Abstracts to Full Publications.

OBJECTIVES: Historically, less than half of peer-reviewed abstracts are published. We set out to determine how many pancreas-related abstracts are published within 5 years of presentation at gastroenterology conferences and to determine a model that predicts successful transition from abstract to journal publication. METHODS: We collected data on study design from all pancreas-related abstracts at the 2010 Digestive Disease Week (DDW), American College of Gastroenterology, and American Pancreatic Association conferences. We then determined whether an abstract was published by October 2015 using a standardized search algorithm. RESULTS: Of 412 abstracts, 39.8% were published. Studies that were of basic science or translational design (P = 0.02, 0.01, respectively); had more listed authors (P = 0.05); employed randomized, prospective, and multicenter methodology (P = 0.02); and were accepted to DDW (P = 0.02) were more likely to be published. After regression, basic/translational studies (P = 0.002, 0.02, respectively) and DDW-accepted abstracts (P = 0.004) continued to predict successful publication. CONCLUSIONS: It is not clear why only 40% of the pancreas abstracts from 2010 were published 5 years later. Some abstracts may go unpublished because of methodological flaws that escape detection during abstract peer review. Therefore, physicians should use caution when applying abstract data to their clinical decision making.

The location of obstruction predicts stent occlusion in malignant gastric outlet obstruction.

BACKGROUND: Gastric outlet obstruction (GOO) can occur with locally invasive or metastatic cancer involving the upper gastrointestinal tract at the pylorus or the duodenum. Endoscopic management with self-expanding metal stents (SEMSs) is often the preferred palliative approach. Stent occlusion is a common reason for failure and reintervention. We set out to determine whether the location of the malignant obstruction is associated with the angulation of the stent and can predict stent occlusion. METHODS: We performed a retrospective review of consecutive patients who underwent successful duodenal stenting with SEMS for malignant GOO between 2006 and 2015 at a large advanced endoscopy referral center. We determined the location of obstruction, the stent angle, and the rate of technical and clinical success of stent placement. We then identified cases of subsequent stent occlusion confirmed by endoscopic evaluation. RESULTS: A total of 100 consecutive patients were included in the study; 91 of these patients had enough data to evaluate SEMS occlusion. A total of 21 patients (23%) developed stent occlusion with a median time of 39 days. The risk of occlusion sequentially increased as the obstruction occurred more distally from the antrum to the third or fourth portion of the duodenum (p = 0.006). This relationship was maintained after controlling for stent angle (p = 0.05). CONCLUSIONS: A distal location of malignant GOO was strongly predictive of stent occlusion, independent of stent angle. This may be due to longer and more complex distal obstructions, along with foreshortening of the stent during placement and tumor infiltration. If replicated, these results will have implications for endoscopic practice and future device development.

An expanded role for mTORC1 in autophagy.

Mechanistic target of rapamycin complex 1 (mTORC1) negatively regulates autophagy at early stages by phosphorylating Unc51-like kinase 1 (ULK1). Our recent study expanded the roles of mTORC1 in autophagy by identifying ultraviolet radiation resistance-associated gene product (UVRAG) as a substrate of mTORC1. This finding has provided new insight into the roles of mTORC1 in cellular membrane processes and cancer.

The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14.

ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation.

Pseudomembranous Collagenous Colitis: A Case of Not-so-Microscopic Colitis.

We present a 72-year-old male who developed progressive, watery diarrhea despite anti-motility agents. On colonoscopy, the mucosa was inflamed and covered with an exudate. Stool studies for Clostridium difficile and Escherichia coli were negative. Biopsies revealed pseudomembranous collagenous colitis, a rare form of microscopic colitis. His symptoms improved dramatically with budesonide therapy.

Hemodynamic Variation and Intravenous Fluids Administered During ERCP and the Association With Post-ERCP Pancreatitis.

OBJECTIVES: We sought to determine whether hypotension and the amount of intravenous (IV) fluids administered during endoscopic retrograde cholangiopancreatography (ERCP) were associated with post-ERCP pancreatitis. METHODS: We identified patients who developed post-ERCP pancreatitis between 2009 and 2013. Using a case-control design, we extracted baseline and intra-ERCP vital signs and the amount of IV fluids given. We used regression to analyze the association between these factors and the risk of post-ERCP pancreatitis. RESULTS: We found no association between intraprocedure hypotension (P = 0.17), bradycardia (P = 0.20), hypoxemia (P = 1.0), dehydration (P = 0.80), and post-ERCP pancreatitis. An increase in mean arterial pressure (MAP) more than 20 units from baseline (odds ratio [OR], 1.8; P = 0.03), increasing amount of IV fluids administered during ERCP (OR, 1.5; P = 0.03), female sex (OR, 2.6; P = 0.001), and younger age (OR, 1.02; P = 0.01) were associated with post-ERCP pancreatitis. In multivariate regression, female sex maintained statistical significance (P = 0.01); MAP more than 20 units from baseline (P = 0.1) and increased IV fluids (P = 0.09) showed an insignificant trend. CONCLUSIONS: Hypotension during ERCP was not associated with post-ERCP pancreatitis. An increase in MAP more than 20 units from baseline and an increase in the amount of IV fluids administered during ERCP may increase the risk of post-ERCP pancreatitis.

A Standardized Assessment of Functional Disability Predicts 1-year Mortality in Patients Undergoing Transjugular Intrahepatic Portosystemic Shunt for Refractory Ascites.

GOALS: To determine the association between functional disability and mortality after transjugular intrahepatic portosystemic shunt (TIPS). BACKGROUND: TIPS is a common therapeutic procedure for cirrhotic patients with refractory ascites. The conventional metric for periprocedure risk stratification is the model for end-stage liver disease (MELD), which uses biochemical parameters to predict post-TIPS mortality. It does not account for functional disability. STUDY: This is a retrospective cohort study of 83 patients admitted at an academic liver transplant center with cirrhosis and refractory ascites for the purpose of TIPS placement. We assessed the association of patients' reported activities of daily living (ADL) on a scale of 1 to 21 before TIPS with a primary outcome of 1-year mortality. Multivariable regression to adjust for MELD and Child class was performed. RESULTS: A higher ADL score or functional independence, was associated with decreased 1-year mortality when modeled as both a continuous variable [odds ratio (OR), 0.80; 95% confidence interval (CI), 0.66-0.97; P=0.02) and a dichotomous variable (ADL 21 vs. <21; OR, 0.21; 95% CI, 0.05-0.70; P=0.01). After adjusting for MELD and Child class, functional independence was associated with decreased 1-year transplant-free mortality (OR, 0.22; 95% CI, 0.05-0.77; P=0.02). An ADL score consistent with dependence (<21) was significantly associated with a 3.40-day (95% CI, 1.76-5.04) longer hospital stay, adjusting for MELD and Child class (P<0.0001). CONCLUSIONS: Functional disability is a predictor of post-TIPS mortality and length of stay after controlling for MELD.

Noninvasive markers in the assessment and management of autoimmune liver diseases.

Historically, liver biopsy has been used to determine the etiology of liver disease, the degree of inflammation, the stage of liver fibrosis, and the response to treatments. In the last decade, the advent of noninvasive tests has improved the diagnosis and management of autoimmune liver diseases. For example, serum markers can identify hepatic inflammation, whereas ultrasound and MRI can diagnose liver fibrosis. Physicians now have a much larger repertoire of diagnostic tests to assess the liver parenchyma compared with liver biopsy alone. In some rare cases, noninvasive tests may provide an alternative to liver biopsy. In general, however, these noninvasive tests complement liver biopsy and provide quick, accurate, and reliable adjunctive data.

Glucose, nitrogen, and phosphate repletion in Saccharomyces cerevisiae: common transcriptional responses to different nutrient signals.

Saccharomyces cerevisiae are able to control growth in response to changes in nutrient availability. The limitation for single macronutrients, including nitrogen (N) and phosphate (P), produces stable arrest in G1/G0. Restoration of the limiting nutrient quickly restores growth. It has been shown that glucose (G) depletion/repletion very rapidly alters the levels of more than 2000 transcripts by at least 2-fold, a large portion of which are involved with either protein production in growth or stress responses in starvation. Although the signals generated by G, N, and P are thought to be quite distinct, we tested the hypothesis that depletion and repletion of any of these three nutrients would affect a common core set of genes as part of a generalized response to conditions that promote growth and quiescence. We found that the response to depletion of G, N, or P produced similar quiescent states with largely similar transcriptomes. As we predicted, repletion of each of the nutrients G, N, or P induced a large (501) common core set of genes and repressed a large (616) common gene set. Each nutrient also produced nutrient-specific transcript changes. The transcriptional responses to each of the three nutrients depended on cAMP and, to a lesser extent, the TOR pathway. All three nutrients stimulated cAMP production within minutes of repletion, and artificially increasing cAMP levels was sufficient to replicate much of the core transcriptional response. The recently identified transceptors Gap1, Mep1, Mep2, and Mep3, as well as Pho84, all played some role in the core transcriptional responses to N or P. As expected, we found some evidence of cross talk between nutrient signals, yet each nutrient sends distinct signals.

Stb3 plays a role in the glucose-induced transition from quiescence to growth in Saccharomyces cerevisiae.

Addition of glucose to quiescent Saccharomyces cerevisiae cells causes the immediate induction of approximately 1000 genes. These genes include ribosomal proteins (RP) and non-RP genes needed for ribosome production and other growth processes. RRPE sequence elements are commonly found 5' of non-RP growth gene ORFs, and Stb3 has recently been identified as an RRPE binding protein. Stb3 overexpression (Stb3OE) produces a slow growth phenotype that is associated with reduced expression of non-RP genes and a drop in the rate of amino acid incorporation. Genes affected by Stb3 are associated with a TGAAAAA motif. Stb3 is restricted to the nucleus in quiescent cells and is immediately released into the cytoplasm after glucose repletion. The Stb3OE slow growth phenotype is reversed by loss of Hos2 histone deactylase activity, consistent with the idea that repression involves histone deacetylation. SCH9 overexpression or PPH22 deletion, mutations that activate target of rapamycin (Tor) nutrient sensing pathways, also reverse the Stb3OE phenotype. Inhibition of Tor signaling makes the phenotype more severe and restricts Stb3 to the nucleus. The results support a model in which Stb3 is one of the components that repress a large set of growth genes as nutrients are depleted. This repression is ended by glucose.