ATG16L1: A multifunctional susceptibility factor in Crohn disease

Genetic variations in the autophagic pathway influence genetic predispositions to Crohn disease. Autophagy, the major lysosomal pathway for degrading and recycling cytoplasmic material, constitutes an important homeostatic cellular process. Of interest, single-nucleotide polymorphisms in ATG16L1 (autophagy-related 16-like 1 [S. cerevisiae]), a key component in the autophagic response to invading pathogens, have been associated with an increased risk of developing Crohn disease. The most common and well-studied genetic variant of ATG16L1 (rs2241880; leading to a T300A conversion) exhibits a strong association with risk for developing Crohn disease. The rs2241880 variant plays a crucial role in pathogen clearance, resulting in imbalanced cytokine production, and is linked to other biological processes, such as the endoplasmic reticulum stress/unfolded protein response. In this review, we focus on the importance of ATG16L1 and its genetic variant (T300A) within the elementary biological processes linked to Crohn disease.     

An alteration in ATG16L1 stability in Crohn disease

Individuals who harbor a common coding polymorphism (Thr300Ala) within a structurally unclassified region of ATG16L1 are at increased risk for the development of Crohn disease. Recently, we reported on the generation and characterization of knockin mice carrying the ATG16L1 T300A variant. We demonstrate that multiple cell types from T300A knock-in mice exhibit reduced selective autophagy, and we mechanistically link this phenotype with an increased susceptibility of ATG16L1 T300A to CASP3- and CASP7-mediated cleavage. These findings demonstrate how a single polymorphism can result in cell type- and pathway-specific disruptions of selective autophagy and alterations in the inflammatory milieu that can contribute to disease.     

An alteration in ATG16L1 stability in Crohn disease

Individuals who harbor a common coding polymorphism (Thr300Ala) within a structurally unclassified region of ATG16L1 are at increased risk for the development of Crohn disease. Recently, we reported on the generation and characterization of knockin mice carrying the ATG16L1 T300A variant. We demonstrate that multiple cell types from T300A knock-in mice exhibit reduced selective autophagy, and we mechanistically link this phenotype with an increased susceptibility of ATG16L1 T300A to CASP3- and CASP7-mediated cleavage. These findings demonstrate how a single polymorphism can result in cell type- and pathway-specific disruptions of selective autophagy and alterations in the inflammatory milieu that can contribute to disease.     

Mechanisms and function of autophagy in intestinal disease

The discovery of numerous genetic variants in the human genome that are associated with inflammatory bowel disease (IBD) has revealed critical pathways that play important roles in intestinal homeostasis. These genetic studies have identified a critical role for macroautophagy/autophagy and more recently, lysosomal function, in maintaining the intestinal barrier and mucosal homeostasis. This review highlights recent work on the functional characterization of IBD-associated human genetic variants in cell type-specific functions for autophagy.     

Unconventional autophagy mediated by the WD40 domain of ATG16L1 is derailed by the T300A Crohn disease risk polymorphism

A coding polymorphism of the critical autophagic effector ATG16L1 (T300A) increases the risk of Crohn disease, but how this mutation influences the function of ATG16L1 has remained unclear. In a recent report, we showed that the A300 allele alters the ability of the C-terminal WD40 domain of ATG16L1 to interact with proteins containing a specific amino acid motif able to recognize this region. This defect impairs the capacity of the motif-containing transmembrane molecule TMEM59 to induce the unconventional autophagic labeling of the same single-membrane vesicles where this protein is located. Such alteration derails the intracellular trafficking of TMEM59 and the xenophagic response against bacterial infection. In contrast, canonical autophagy remains unaffected in the presence of ATG16L1^T300A. These data argue that the T300A polymorphism impairs the unconventional autophagic activities carried out by the WD40 domain, a region of ATG16L1 whose function has remained poorly understood.     

Genetic association and functional role of Crohn disease risk alleles involved in microbial sensing,autophagy, and endoplasmic reticulum (ER) stress

Genome-wide association studies have identified several genes implicated in autophagy (ATG16L1, IRGM, ULK1, LRRK2, and MTMR3), intracellular bacterial sensing (NOD2), and endoplasmic reticulum (ER) stress (XBP1 and ORMDL3) to be associated with Crohn disease (CD). We studied the known CD-associated variants in these genes in a large cohort of 3451 individuals (1744 CD patients, 793 ulcerative colitis (UC) patients and 914 healthy controls). We also investigated the functional phenotype linked to these genetic variants. Association with CD was confirmed for NOD2, ATG16L1, IRGM, MTMR3, and ORMDL3. The risk for developing CD increased with an increasing number of risk alleles for these genes (P < 0.001, OR 1.26 [1.20 to 1.32]). Three times as many (34.8%) CD patients carried a risk allele in all three pathways, in contrast to 13.3% of the controls (P < 0.0001, OR = 3.46 [2.77 to 4.32]). For UC, no significant association for one single nucleotide polymorphism (SNP) was found, but the risk for development of UC increased with an increasing total number of risk alleles (P = 0.001, OR = 1.10 [1.04 to 1.17]). We found a genetic interaction between reference SNP (rs)2241880 (ATG16L1) and rs10065172 (IRGM) in CD. Functional experiments hinted toward an association between an increased genetic risk and an augmented inflammatory status, highlighting the relevance of the genetic findings.     

Silencing of EEF2K (eukaryotic elongation factor-2 kinase) reveals AMPK-ULK1-dependent autophagy in colon cancer cells

EEF2K (eukaryotic elongation factor-2 kinase), also known as Ca²⁺/calmodulin-dependent protein kinase III, functions in downregulating peptide chain elongation through inactivation of EEF2 (eukaryotic translation elongation factor 2). Currently, there is a limited amount of information on the promotion of autophagic survival by EEF2K in breast and glioblastoma cell lines. However, the precise role of EEF2K in carcinogenesis as well as the underlying mechanism involved is still poorly understood. In this study, contrary to the reported autophagy-promoting activity of EEF2K in certain cancer cells, EEF2K is shown to negatively regulate autophagy in human colon cancer cells as indicated by the increase of LC3-II levels, the accumulation of LC3 dots per cell, and the promotion of autophagic flux in EEF2K knockdown cells. EEF2K negatively regulates cell viability, clonogenicity, cell proliferation, and cell size in colon cancer cells. Autophagy induced by EEF2K silencing promotes cell survival and does not potentiate the anticancer efficacy of the AKT inhibitor MK-2206. In addition, autophagy induced by silencing of EEF2K is attributed to induction of protein synthesis and activation of the AMPK-ULK1 pathway, independent of the suppression of MTOR activity and ROS generation. Knockdown of AMPK or ULK1 significantly abrogates EEF2K silencing-induced increase of LC3-II levels, accumulation of LC3 dots per cell as well as cell proliferation in colon cancer cells. In conclusion, silencing of EEF2K promotes autophagic survival via activation of the AMPK-ULK1 pathway in colon cancer cells. This finding suggests that upregulation of EEF2K activity may constitute a novel approach for the treatment of human colon cancer.     

Induction of autophagy through CLEC4E in combination with TLR4: an innovative strategy to restrict the survival of Mycobacterium tuberculosis

ABSTRACT

Host-directed therapies are gaining considerable impetus because of the emergence of drug-resistant strains of pathogens due to antibiotic therapy. Therefore, there is an urgent need to exploit alternative and novel strategies directed at host molecules to successfully restrict infections. The C-type lectin receptor CLEC4E and Toll-like receptor TLR4 expressed by host cells are among the first line of defense in encountering pathogens. Therefore, we exploited signaling of macrophages through CLEC4E in association with TLR4 agonists (C₄.T₄) to control the growth of Mycobacterium tuberculosis (Mtb). We observed significant improvement in host immunity and reduced bacterial load in the lungs of Mtb-infected mice and guinea pigs treated with C₄.T₄ agonists. Further, intracellular killing of Mtb was achieved with a 10-fold lower dose of isoniazid or rifampicin in conjunction with C₄.T₄ than the drugs alone. C₄.T₄ activated MYD88, PtdIns3K, STAT1 and RELA/NFKB, increased lysosome biogenesis, decreased Il10 and Il4 gene expression and enhanced macroautophagy/autophagy. Macrophages from autophagy-deficient (atg5 knockout or Becn1 knockdown) mice showed elevated survival of Mtb. The present findings also unveiled the novel role of CLEC4E in inducing autophagy through MYD88, which is required for control of Mtb growth. This study suggests a unique immunotherapeutic approach involving CLEC4E in conjunction with TLR4 to restrict the survival of Mtb through autophagy.     

IL17A augments autophagy in Mycobacterium tuberculosis-infected monocytes from patients with active tuberculosis in association with the severity of the disease

During mycobacterial infection, macroautophagy/autophagy, a process modulated by cytokines, is essential for mounting successful host responses. Autophagy collaborates with human immune responses against Mycobacterium tuberculosis (Mt) in association with specific IFNG secreted against the pathogen. However, IFNG alone is not sufficient to the complete bacterial eradication, and other cytokines might be required. Actually, induction of Th1 and Th17 immune responses are required for protection against Mt. Accordingly, we showed that IL17A and IFNG expression in lymphocytes from tuberculosis patients correlates with disease severity. Here we investigate the role of IFNG and IL17A during autophagy in monocytes infected with Mt H37Rv or the mutant MtΔRD1. Patients with active disease were classified as high responder (HR) or low responder (LR) according to their T cell responses against Mt. IL17A augmented autophagy in infected monocytes from HR patients through a mechanism that activated MAPK1/ERK2-MAPK3/ERK1 but, during infection of monocytes from LR patients, IL17A had no effect on the autophagic response. In contrast, addition of IFNG to infected monocytes, increased autophagy by activating MAPK14/p38 α both in HR and LR patients. Interestingly, proteins codified in the RD1 region did not interfere with IFNG and IL17A autophagy induction. Therefore, in severe tuberculosis patients' monocytes, IL17A was unable to augment autophagy because of a defect in the MAPK1/3 signaling pathway. In contrast, both IFNG and IL17A increased autophagy levels in patients with strong immunity to Mt, promoting mycobacterial killing. Our findings might contribute to recognize new targets for the development of novel therapeutic tools to fight the pathogen.     

Secretory autophagy of lysozyme in Paneth cells

Secretion of antimicrobial proteins is an important host defense mechanism against bacteria, yet how secretory cells maintain function during bacterial invasion has been unclear. We discovered that Paneth cells, specialized secretory cells in the small intestine, react to bacterial invasion by rerouting a critical secreted antibacterial protein through a macroautophagy/autophagy-based secretion system termed secretory autophagy. Mice harboring a mutation in an essential autophagy gene, a mutation which is common in Crohn disease patients, cannot reroute their antimicrobial cargo during bacterial invasion and thus have compromised innate immunity. We showed that this alternative secretion system is triggered by both a cell-intrinsic mechanism, involving the ER stress response, and a cell-extrinsic mechanism, involving subepithelial innate immune cells. Our findings uncover a new role for secretory autophagy in host defense and suggest how a mutation in an autophagy gene can predispose individuals to Crohn disease.     

The multi-step mechanism and biological role of noncanonical autophagy targeting Streptococcus pneumoniae during the early stages of infection

ABSTRACT

Multiple autophagic processes are triggered in response to bacterial infection as the host attempts to eliminate intracellular invaders. However, it is still unclear how the mechanisms contributing to canonical macroautophagy/autophagy, including xenophagy, coordinate with the more recently described features that are characteristic of noncanonical autophagy. Recently, we revealed that infection with Streptococcus pneumoniae can trigger the formation of RB1CC1/FIP200-independent LC3-associated phagosome-like vacuoles (PcLVs) that contain the pneumococci at an early stage of infection. We also found that interactions of SQSTM1/p62 with the ATG16L1 WD domain are essential for PcLV formation. Intriguingly, PcLVs were required for the subsequent generation of bactericidal autophagic vacuoles (PcAVs). Furthermore, we also identified LC3-delocalized SQSTM1-positive PcLVs as intracellular intermediates that link PcLVs and PcAVs. These findings reveal a novel multi-step mechanism that contributes to xenophagy of the critical S. pneumoniae respiratory pathogen.     

ATG16L1 phosphorylation is oppositely regulated by CSNK2/casein kinase 2 and PPP1/protein phosphatase 1 which determines the fate of cardiomyocytes during hypoxia/reoxygenation

Recent studies have shown that the phosphorylation and dephosphorylation of ULK1 and ATG13 are related to autophagy activity. Although ATG16L1 is absolutely required for autophagy induction by affecting the formation of autophagosomes, the post-translational modification of ATG16L1 remains elusive. Here, we explored the regulatory mechanism and role of ATG16L1 phosphorylation for autophagy induction in cardiomyocytes. We showed that ATG16L1 was a phosphoprotein, because phosphorylation of ATG16L1 was detected in rat cardiomyocytes during hypoxia/reoxygenation (H/R). We not only demonstrated that CSNK2 (casein kinase 2) phosphorylated ATG16L1, but also identified the highly conserved Ser139 as the critical phosphorylation residue for CSNK2. We further established that ATG16L1 associated with the ATG12-ATG5 complex in a Ser139 phosphorylation-dependent manner. In agreement with this finding, CSNK2 inhibitor disrupted the ATG12-ATG5-ATG16L1 complex. Importantly, phosphorylation of ATG16L1 on Ser139 was responsible for H/R-induced autophagy in cardiomyocytes, which protects cardiomyocytes from apoptosis. Conversely, we determined that wild-type PPP1 (protein phosphatase 1), but not the inactive mutant, associated with ATG16L1 and antagonized CSNK2-mediated phosphorylation of ATG16L1. Interestingly, one RVxF consensus site for PPP1 binding in the C-terminal tail of ATG16L1 was identified; mutation of this site disrupted its association with ATG16L1. Notably, CSNK2 also associated with PPP1, but ATG16L1 depletion impaired the interaction between CSNK2 and PPP1. Collectively, these data identify ATG16L1 as a bona fide physiological CSNK2 and PPP1 substrate, which reveals a novel molecular link from CSNK2 to activation of the autophagy-specific ATG12-ATG5-ATG16L1 complex and autophagy induction.     

Crosstalk between lysine methylation and phosphorylation of ATG16L1 dictates the apoptosis of hypoxia/reoxygenation-induced cardiomyocytes

Post-translational modifications of autophagy-related (ATG) genes are necessary to modulate their functions. However, ATG protein methylation and its physiological role have not yet been elucidated. The methylation of non-histone proteins by SETD7, a SET domain-containing lysine methyltransferase, is a novel regulatory mechanism to control cell protein function in response to various cellular stresses. Here we present evidence that the precise activity of ATG16L1 protein in hypoxia/reoxygenation (H/R)-treated cardiomyocytes is regulated by a balanced methylation and phosphorylation switch. We first show that H/R promotes autophagy and decreases SETD7 expression, whereas autophagy inhibition by 3-MA increases SETD7 level in cardiomyocytes, implying a tight correlation between autophagy and SETD7. Then we demonstrate that SETD7 methylates ATG16L1 at lysine 151 while KDM1A/LSD1 (lysine demethylase 1A) removes this methyl mark. Furthermore, we validate that this methylation at lysine 151 impairs the binding of ATG16L1 to the ATG12–ATG5 conjugate, leading to inhibition of autophagy and increased apoptosis in H/R-treated cardiomyocytes. However, the cardiomyocytes with shRNA-knocked down SETD7 or inhibition of SETD7 activity by a small molecule chemical, display increased autophagy and decreased apoptosis following H/R treatment. Additionally, methylation at lysine 151 inhibits phosphorylation of ATG16L1 at S139 by CSNK2 which was previously shown to be critical for autophagy maintenance, and vice versa. Together, our findings define a novel modification of ATG16L1 and highlight the importance of an ATG16L1 phosphorylation-methylation switch in determining the fate of H/R-treated cardiomyocytes.     

Up‐regulated SAMD9L modulated by TLR2 and HIF‐1α as a promising biomarker in tuberculosis

The aim of this study was to identify potential biomarkers of TB in blood and determine their function in Mtb‐infected macrophages. First of all, WGCNA was used to analyse 9451 genes with significant changes in TB patients’ whole blood. The 220 interferon‐γ‐related genes were identified, and then 30 key genes were screened using Cytoscape. Then, the AUC values of key genes were calculated to further narrow the gene range. Finally, we identified 9 genes from {"type":"entrez-geo","attrs":{"text":"GSE19444","term_id":"19444"}}GSE19444. ROC analysis showed that SAMD9L, among 9 genes, had a high diagnostic value (AUC = 0.925) and a differential diagnostic value (AUC>0.865). To further narrow down the range of DEGs, the top 10 hub‐connecting genes were screened from monocytes ({"type":"entrez-geo","attrs":{"text":"GSE19443","term_id":"19443"}}GSE19443). Finally, we obtained 4 genes (SAMD9L, GBP1, GBP5 and STAT1) by intersections of genes from monocytes and whole blood. Among them, it was found that the function of SAMD9L was unknown after data review, so this paper studied this gene. Our results showed that SAMD9L is up‐regulated and suppresses cell necrosis, and might be regulated by TLR2 and HIF‐1α during Mtb infection. In addition, miR‐181b‐5p is significantly up‐regulated in the peripheral blood plasma of tuberculosis patients, which has a high diagnostic value (AUC = 0.969).     

Polymorphisms of the SLC11A1 gene and resistance to bovine tuberculosis in African Zebu cattle

Bovine tuberculosis (BTB) is a considerable health threat to livestock keepers and general communities in many developing countries. Information on genetic resistance or susceptibility because of polymorphisms of candidate genes could be used in making selection decisions for breeding disease tolerant/resistant animals. Here, we investigated associations between polymorphisms at the solute carrier family 11 (proton‐coupled divalent metal ion transporters), member 1 gene (SLC11A1, previously known as natural resistant associated macrophage protein 1, NRAMP1), with BTB phenotypes in Chadian cattle. Phenotypes were (i) single intradermal comparative cervical tuberculin test (SICCT) outcome, (ii) presence of gross visible lung lesions, (iii) a bacteriological culture test outcome and (iv) a predicted true BTB infection status using a Bayesian model. All traits were recorded as binary (presence or absence) traits. A total of 211 cattle were genotyped for a microsatellite within the SLC11A1 candidate gene. Standard linear and threshold‐liability models regressing BTB traits on copy number of SLC11A1 alleles revealed statistically significant effects of SLC11A1 alleles (P < 0.001) on most BTB traits. Polymorphisms (alleles 211, 215 and 217) are significantly related to lower incidence of BTB traits in Chadian cattle. This is the first study to report the association of SLC11A1 gene polymorphisms with BTB traits in Chadian or any other African cattle breeds.     

Crystal structures of Mycobacterium tuberculosis S-adenosyl-L-homocysteine hydrolase in ternary complex with substrate and inhibitors

S-adenosylhomocysteine hydrolase (SAHH) is a ubiquitous enzyme that plays a central role in methylation-based processes by maintaining the intracellular balance between S-adenosylhomocysteine (SAH) and S-adenosylmethionine. We report the first prokaryotic crystal structure of SAHH, from Mycobacterium tuberculosis (Mtb), in complex with adenosine (ADO) and nicotinamide adenine dinucleotide. Structures of complexes with three inhibitors are also reported: 3'-keto aristeromycin (ARI), 2-fluoroadenosine, and 3-deazaadenosine. The ARI complex is the first reported structure of SAHH complexed with this inhibitor, and confirms the oxidation of the 3' hydroxyl to a planar keto group, consistent with its prediction as a mechanism-based inhibitor. We demonstrate the in vivo enzyme inhibition activity of the three inhibitors and also show that 2-fluoradenosine has bactericidal activity. While most of the residues lining the ADO-binding pocket are identical between Mtb and human SAHH, less is known about the binding mode of the homocysteine (HCY) appendage of the full substrate. We report the 2.0 A resolution structure of the complex of SAHH cocrystallized with SAH. The most striking change in the structure is that binding of HCY forces a rotation of His363 around the backbone to flip out of contact with the 5' hydroxyl of the ADO and opens access to a nearby channel that leads to the surface. This complex suggests that His363 acts as a switch that opens up to permit binding of substrate, then closes down after release of the cleaved HCY. Differences in the entrance to this access channel between human and Mtb SAHH are identified.     

Evidence of expression variation and allelic imbalance in Crohn's disease susceptibility genes NOD2 and ATG16L1 in human dendritic cells

Human dendritic cells (DCs) play an important role in induction and progression of Crohn's disease (CD). Accumulating evidence suggests that viral infection is required to trigger CD pathogenesis in genetically predisposed individuals. NOD2 and ATG16L1 are among the major CD susceptibility genes implicated in impaired immune response to bacterial infection. In this study, we investigated gene expression and allelic imbalance (AI) of NOD2 and ATG16L1 using common variants in human monocyte-derived DCs. Significant AI was observed in ~ 40% and ~ 70% of NOD2 and ATG16L1 heterozygotes, respectively (p < 0.05). AI of NOD2 was inversely associated with its expression level (p = 0.015). No correlation was detected between gene expression and AI for ATG16L1. When infected with Newcastle Disease Virus (NDV), NOD2 expression in DCs was induced about four-fold (p < 0.001), whereas ATG16L1 expression was not affected (p = 0.88). In addition, NDV infection tended to lower the variance in AI among DC populations for the NOD2 gene (p = 0.05), but not the ATG16L1 gene (p = 0.32). Findings of a simulation study, aimed to verify whether the observed variation in gene expression and AI is a result of sample-to-sample variability or experimental measurement error, suggested that NOD2 AI is likely to result from a deterministic event at a single cell level. Overall, our results present initial evidence that AI of the NOD2 and ATG16L1 genes exists in populations of human DCs. In addition, our findings suggest that viral infection may regulate NOD2 expression.     

Piperlongumine restores the balance of autophagy and apoptosis by increasing BCL2 phosphorylation in rotenone-induced Parkinson disease models

Parkinson disease (PD) is the second most common neurodegenerative disorder after Alzheimer disease and is caused by genetics, environmental factors and aging, with few treatments currently available. Apoptosis and macroautophagy/autophagy play critical roles in PD pathogenesis; as such, modulating their balance is a potential treatment strategy. BCL2 (B cell leukemia/lymphoma 2) is a key molecule regulating this balance. Piperlongumine (PLG) is an alkaloid extracted from Piper longum L. that has antiinflammatory and anticancer effects. The present study investigated the protective effects of PLG in rotenone-induced PD cell and mouse models. We found that PLG administration (2 and 4 mg/kg) for 4 wk attenuated motor deficits in mice and prevented the loss of dopaminergic neurons in the substantia nigra induced by oral administration of rotenone (10 mg/kg) for 6 wk. PLG improved cell viability and enhanced mitochondrial function in primary neurons and SK-N-SH cells. These protective effects were exerted via inhibition of apoptosis and induction of autophagy through enhancement of BCL2 phosphorylation at Ser70. These results demonstrate that PLG exerts therapeutic effects in a rotenone-induced PD models by restoring the balance between apoptosis and autophagy.

Abbreviations: 6-OHDA, 6-hydroxydopamine; ACTB, actin, beta; BafA1, bafilomycin A₁; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BCL2, B cell leukemia/lymphoma2; BECN1, Beclin 1, autophagy related; CoQ10, coenzyme Q₁₀; COX4I1/COX IV, cytochrome c oxidase subunit 4I1; CsA, cyclosporine A; ED50, 50% effective dose; FITC, fluorescein isothiocyanate; GFP, green fluorescent protein; HPLC, high-performance liquid chromatography; JC-1, tetraethylbenz-imidazolylcarbocyanine iodide; LC3, microtubule-associated protein 1 light chain3; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LDH, lactate dehydrogenase; l-dopa, 3, 4-dihydroxyphenyl-l-alanine; MAPK8/JNK1, mitogen-activated protein kinase 8; MMP, mitochondrial membrane potential; mPTP, mitochondrial permeability transition pore; mRFP, monomeric red fluorescent protein; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NFE2L2/NRF2, nuclear factor, erythroid derived 2, like 2; PD, Parkinson disease; PLG, piperlongumine; pNA, p-nitroanilide; PI, propidium iodide; PtdIns3K, phosphatidylinositol 3-kinase; PtdIns3P, phosphatidylinositol-3-phosphate; PTX, paclitaxel; Rap, rapamycin; SQSTM1/p62, sequestosome 1; TH, tyrosine hydroxylase; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; WIPI2, WD repeat domain, phosphoinositide interacting 2; ZFYVE1/DFCP1, zinc finger, FYVE domain containing 1.