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Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We identify six ULK‐dependent phosphorylation sites on VPS15, mutation of which reduces autophagosome formation in cells and VPS34 activity in vitro. Mutation of serine 861, the major VPS15 phosphosite, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells reveals two novel ULK‐dependent phenotypes downstream of VPS15 removal that can be partially recapitulated by chronic VPS34 inhibition, starvation‐independent accumulation of ULK substrates and kinase activity‐regulated recruitment of autophagy proteins to ubiquitin‐positive structures. SYNOPSIS: Serine/threonine kinase ULK is the sole protein kinase in the autophagic signalling cascade and a key autophagy regulator. This study identifies the VPS34 complex component VPS15 as a novel ULK substrate in mammalian cells, and reveals a role for ULK‐VPS15 signalling in autophagosome formation. Unbiased phosphoproteomics and in vitro kinase assays identify several novel ULK substrates.ULK phosphorylates VPS15 and PRKAG2, components of VPS34 and AMPK complexes, respectively.The ULK‐VPS15 signalling axis regulates VPS34 complex activity in vitro, and autophagy in cells.VPS15 knockout cells accumulate subsets of ULK phospho‐substrates.Upon VPS15 loss, autophagy proteins redistribute to ubiquitin‐positive condensates in an ULK‐ and VPS34‐dependent manner. [ABSTRACT FROM AUTHOR]

Background and Aim: Liver disease mortality rates continue to rise due to late diagnosis. We need noninvasive tests to be made available in the community that can identify patients at risk from a serious liver‐related event (SLE). We examine the performance of a blood test, the liver traffic light test (LTLT), with regard to its ability to predict survival and SLEs. Methods: Using routinely gathered clinical data, sequential LTLT test results from 4854 individuals with suspected liver disease were prospectively analyzed (median follow‐up 41 months). An SLE was defined as the development of cirrhosis, liver failure, ascites, or varices. Patients were graded as follows: red (high risk), amber (intermediate risk), and green (low risk). Results: Overall, 565 individuals experienced an SLE (11.6%). The area under the curve (AUC) for the continuous LTLT variable was 0.87 (95% confidence interval 0.85–0.89) for prediction of an SLE and 0.81 (0.78–0.84) for mortality. When categorized into red/amber/green grades, a red LTLT result predicted an SLE with negative and positive predictive values of 0.97 and 0.29, respectively. A red LTLT score predicted mortality with negative and positive predictive values of 0.98 and 0.18, respectively. Kaplan–Meier plots demonstrated increased mortality and SLEs in the red group versus the green and amber groups (P < 0.001) and an increase in SLEs in the amber versus green group (P < 0.001). Conclusion: Here, the LTLT is further validated for the prediction of survival and SLE development. The LTLT could aid primary care risk management and referral pathways with the aim of detecting and treating liver disease earlier in the general population. [ABSTRACT FROM AUTHOR]

Background: Mitochondria play a fundamental role in the pathogenesis of alcoholic liver disease (ALD). The preservation of functional mitochondria during toxic alcohol insults is essential for cell survival and is maintained by key processes known as mitochondrial dynamics, including fragmentation and fusion, which are regulated by mitochondria‐shaping proteins (MSP). We have shown mitochondrial dynamics to be distorted by alcohol in cellular and animal models, but the effect in humans remains unknown. Methods: Hepatic gene expression of the main MSP involved in the mitochondrial fusion and fragmentation pathways was evaluated in patients with alcoholic hepatitis (AH) by DNA microarray (n = 15) and Reverse Transcription Polymerase Chain Reaction (n = 32). The activation of dynamin‐1‐like protein (Drp1) was also investigated in mitochondria isolated from liver biopsies of ALD patients (n = 8). The effects of alcohol on mitochondrial dynamics and on MSP protein expression were studied in human precision‐cut liver slices (PCLS) exposed for 24 hours to increasing doses of ethanol (EtOH; 50 to 250 mM). Results: A profound hyperactivation of the fragmentation pathway was observed in AH patients, with a significant increase in the expression of Drp1 and its adapters/receptors. The translocation of Drp1 to the mitochondria was also induced in patients with severe ALD and was affected in the PCLS with short‐term exposure to EtOH but only mildly. The fusion pathway was not altered in ALD, and this was confirmed in the PCLS model. Conclusions: The present study reveals the role of mitochondrial dynamics in human ALD, confirming our previous observations in animal and cell culture models of ALD. Taken together, we show that alcohol has a significant impact on the fragmentation pathway, and we confirm Drp1 as a potential therapeutic target in severe ALD. [ABSTRACT FROM AUTHOR]

Background & Aims: Dietary changes can modulate gut microbiota and interact with cirrhosis. Our prior study demonstrated that microbial diversity was higher in cirrhotics from Turkish vs the USA, which was associated with lower risk of 90‐day hospitalizations. We aimed to define gut microbial functional and metabolomic changes to increase insight into benefits of the Mediterranean compared to Western diets. Methods: In all, 139 Turkish (46 controls/50 compensated/43 decompensated) and 157 American subjects (48 controls/59 compensated/50 decompensated) were studied. Turkish subjects consumed a modified Mediterranean diet with daily fermented milk intake, whereas Americans consumed a Western diet. Predicted gut microbial functionalities and plasma metabolomics were compared between/within countries. Correlation network differences between microbiota and metabolites in cirrhotics from Turkey vs the USA were evaluated. Results: Predicted microbial function showed lower amino acid, bioenergetics and lipid pathways, with functions related to vitamin B, glycan, xenobiotic metabolism, DNA/RNA synthesis, in cirrhotics from Turkey compared to the USA. Plasma metabolomics demonstrated higher relative lactate levels in Turkey vs the USA. The metabolite changes in decompensated cirrhosis, compared to controls, showed similar trends in Turkey and the USA, with reduced lipids and phosphocholines. Phosphocholines were significantly lower in patients hospitalized in 90 days (P =.03). Correlation networks in cirrhotics demonstrated linkage differences between beneficial taxa, Blautia and Oscillispira, and lactate and unsaturated lipids, in Turkey compared to American patients. Conclusions: A modified Mediterranean diet was associated with altered plasma metabolomics and beneficially alters microbiota functionality and correlations compared to Western diet in cirrhosis. These altered diet‐microbial interactions could potentially affect the 90‐day hospitalization risk. [ABSTRACT FROM AUTHOR]

The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X-ray crystallography has been a powerful approach to understand protein-protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX-MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX-MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX-MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ-Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein-protein complexes, and is an excellent strategy to optimize constructs for high-resolution structural approaches. [ABSTRACT FROM AUTHOR]

The importance of epigenetic changes in the development of hepatic steatosis is largely unknown. The histone variantmacroH2A1 under alternative splicing gives rise tomacroH2A1.1 andmacroH2A1.2. In this study, we show that the macroH2A1 isoforms play an important role in the regulation of lipid accumulation in hepatocytes. Hepatoma cell line and immortalized human hepatocytes transiently transfected or knocked down with macroH2A1 isoforms were used as in vitro model of fat-induced steatosis. Gene expressions were analyzed by quantitative PCR array and Western blot. Chromatin immunoprecipitation analysis was performed to check the association of histone H3 lysine 27 trimethylation (H3K27me3) and histone H3 lysine 4 trimethylation (H3K4me3) with the promoter of lipogenic genes. Livers from knockout mice that are resistant to lipid deposition despite a high-fat diet were used for histopathology. We found that macroH2A1.2 is regulated by fat uptake and that its overexpression caused an increase in lipid uptake, triglycerides, and lipogenic genes compared with macroH2A1.1. This suggests that macroH2A1.2 is important for lipid uptake, whereas macroH2A1.1 was found to be protective. The result was supported by a high positivity for macroH2A1.1 in knockout mice for genes targeted by macroH2A1 (Atp5a1 and Fam73b), that under a high-fat diet presented minimal lipidosis. Moreover, macroH2A1 isoforms differentially regulate the expression of lipogenic genes by modulating the association of the active (H3K4me3) and repressive (H3K27me3) histonemarks on their promoters. This study underlines the importance of the replacement of non-canonical histones in the regulation of genes involved in lipid metabolism in the progression of steatosis. [ABSTRACT FROM AUTHOR]

Background & Aims Variations in intestinal microbiota may influence acetaminophen metabolism. This study aimed to determine whether intestinal microbiota are a source of differential susceptibility to acetaminophen-induced hepatotoxicity. Methods Conventionally housed C3H/HeH ( CH) and C3H/HeH germ-free ( GF) mice were administered a 200 mg/kg IP dose of acetaminophen. The severity of hepatotoxicity at 8 h was assessed by histology and biochemical indices. A urinary metabolic profile was obtained using 1H- NMR. Baseline hepatic glutathione content and CYP2E1 expression were quantified. An additional group of C3H/HeJ ( LPS-r) mice were assessed to determine the contribution of LPS/ TLR4 signalling. Results Baseline glutathione levels were significantly reduced ( P = 0.03) in GF mice. CYP2E1 mRNA expression and protein levels were not altered. Interindividual variability did not differ between GF and CH groups. No significant differences in the extent of hepatocellular injury (ALT or percentage necrosis) were demonstrated. However, a milder acute liver failure (ALF) phenotype was shown in GF compared with CH mice, with reduced plasma bilirubin and creatinine and increased blood glucose. Differential acetaminophen metabolism was demonstrated. GF mice displayed a higher urinary acetaminophen-sulphate:glucuronide ratio compared with CH ( P = 0.01). Urinary analysis showed metabolic differentiation of GF and CH groups at baseline and 8 h (cross-validated anova P = 1 × 10-22). Interruption of TLR4 signalling in LPS-r mice had additional protective effects. Conclusion Variations in intestinal microbiota do not fully explain differential susceptibility to acetaminophen-induced hepatotoxicity. GF mice experienced some protection from secondary complications following acetaminophen overdose and this may be mediated through reduced TLR4/ LPS signalling. [ABSTRACT FROM AUTHOR]

Highlights: [•] The structure of CTNNBL1 includes an abbreviated armadillo domain. [•] CTNNBL1 is a novel NLS binding protein with a unique carboxy-terminal structure. [•] Structure-based mutagenesis of CTNNBL1 shows NLS binding distinct from karyopherins. [Copyright &y& Elsevier]

Cell-mediated immune responses play an important role in the control of tumour growth. CD4 and CD8 T cells recognise tumour antigens presented via major histocompatibility complex molecules of antigen presenting cells and develop into effector cells with the ability to identify and kill tumour cells. Here, we re-examine the adaptive immune response to tumour antigens expressed by hepatocellular carcinoma (HCC) and discuss approaches that could be applied in future T-cell-based immunotherapy schedules to induce a potent and effective antitumour immunity. Moreover, we discuss cytotoxic T lymphocyte and Th1 responses to tumour antigens in patients with HCC and evaluate the effects of conventional treatments on antitumour T-cell responses. [ABSTRACT FROM AUTHOR]

The article presents a commentary on the approach of Alexis de Tocqueville on religion. The author explores secularization, separation of church and state, Christianity, collapse of morale, and theological deductions to define Tocqueville's claims concerning religion. He also comments on Tocqueville's view of politics.

KIF16B is a newly identified kinesin that regulates the intracellular motility of early endosomes. KIF16B is unique among kinesins in that its cargo binding is mediated primarily by the strong interaction of its PX domain with endosomal lipids. To elucidate the structural basis of this unique endosomal anchoring activity of KIF16B-PX, we determined the crystal structure of the PX domain and performed in vitro and cellular membrane binding measurements for KIF16B-PX and mutants. The most salient structural feature of KIF16B-PX is that two neighboring residues, L1248 and F1249, on the membrane-binding surface form a protruding hydrophobic stalk with a large solvent-accessible surface area. This unique structure, arising from the complementary stacking of the two side chains and the local conformation, allows strong hydrophobic membrane interactions and endosome tethering. The presence of similar hydrophobic pairs in the amino-acid sequences of other membrane-binding domains and proteins suggests that the same structural motif may be shared by other membrane-binding proteins, whose physiological functions depend on strong hydrophobic membrane interactions. [ABSTRACT FROM AUTHOR]

Many disease-related processes occur via protein complexes that are considered undruggable with small molecules. An example is RAS, which is frequently mutated in cancer and contributes to initiation and maintenance of the disease by constitutive signal transduction through protein interaction with effector proteins, like PI3K, RAF and RALGDS. Such protein interactions are therefore significant targets for therapy. We describe a single immunoglobulin variable region domain that specifically binds to activated GTP-bound RAS and prevents RAS-dependent tumorigenesis in a mouse model. The crystal structure of the immunoglobulin–RAS complex shows that the variable region competitively binds to the conformationally variant regions of RAS, where its signalling effector molecules interact. This allows the plasma membrane targeted single domain intrabody to inhibit signalling by mutant RAS. This mode of action is a novel advance to directly interfere with oncogenic RAS function in human cancer and shows a universally applicable approach to develop macromolecules to combat cancer. In addition, this method illustrates a general means for interfering with protein interactions that are commonly considered intractable as conventional drug targets. [ABSTRACT FROM AUTHOR]

The article focuses on the life and works of Chrétien-Guillaume de Lamoignon de Malesherbes, a magistrate and critic of royal absolutism in France. Although Malesherbes was an expert botanist and agronomist, he remain unknown since he was not a publishing scholar. He gained his botanical expertise concerning the natural classification of plants from his mentor Bernard de Jussieu. Moreover, it discusses the reasons why his proposals did not survive with the advent of the French Revolution.

ESCRT (endosomal sorting complex required for transport) complexes orchestrate efficient sorting of ubiquitinated transmembrane receptors to lysosomes via multivesicular bodies (MVBs). Yeast ESCRT-I and ESCRT-II interact directly in vitro; however, this association is not detected in yeast cytosol. To gain understanding of the molecular mechanisms of this link, we have characterised the ESCRT-I/-II supercomplex and determined the crystal structure of its interface. The link is formed by the vacuolar protein sorting (Vps)28 C-terminus (ESCRT-I) binding with nanomolar affinity to the Vps36-NZF-N zinc-finger domain (ESCRT-II). A hydrophobic patch on the Vps28-CT four-helix bundle contacts the hydrophobic knuckles of Vps36-NZF-N. Mutation of the ESCRT-I/-II link results in a cargo-sorting defect in yeast. Interestingly, the two Vps36 NZF domains, NZF-N and NZF-C, despite having the same core fold, use distinct surfaces to bind ESCRT-I or ubiquitinated cargo. We also show that a new component of ESCRT-I, Mvb12 (YGR206W), engages ESCRT-I directly with nanomolar affinity to form a 1:1:1:1 heterotetramer. Mvb12 does not affect the affinity of ESCRT-I for ESCRT-II in vitro. Our data suggest a complex regulatory mechanism for the ESCRT-I/-II link in yeast. [ABSTRACT FROM AUTHOR]

The article talks about the characteristics of Alexis de Tocqueville, the liberal who has created "Democracy in America" and "The Old Régime and the French Revolution." These two major works of his were well-received in his lifetime. However, an analysis of his correspondence dictate that along with his rigidity and inability to compromise, he was temperamentally unsuited to political or administrative life, a character found to be similar with that of his great grandfather Malesherbes.