The V-ATPase/ATG16L1 axis is controlled by the V1H subunit.

Defects in organellar acidification indicate compromised or infected compartments. Recruitment of the autophagy-related ATG16L1 complex to pathologically neutralized organelles targets ubiquitin-like ATG8 molecules to perturbed membranes. How this process is coupled to proton gradient disruption is unclear. Here, we reveal that the V1H subunit of the vacuolar ATPase (V-ATPase) proton pump binds directly to ATG16L1. The V1H/ATG16L1 interaction only occurs within fully assembled V-ATPases, allowing ATG16L1 recruitment to be coupled to increased V-ATPase assembly following organelle neutralization. Cells lacking V1H fail to target ATG8s during influenza infection or after activation of the immune receptor stimulator of interferon genes (STING). We identify a loop within V1H that mediates ATG16L1 binding. A neuronal V1H isoform lacks this loop and is associated with attenuated ATG8 targeting in response to ionophores in primary murine and human iPSC-derived neurons. Thus, V1H controls ATG16L1 recruitment following proton gradient dissipation, suggesting that the V-ATPase acts as a cell-intrinsic damage sensor.

Non-autophagic Golgi-LC3 lipidation facilitates TFE3 stress response against Golgi dysfunction.

Lipidated ATG8/LC3 proteins are recruited to single membrane compartments as well as autophagosomes, supporting their functions. Although recent studies have shown that Golgi-LC3 lipidation follows Golgi damage, its molecular mechanism and function under Golgi stress remain unknown. Here, by combining DLK1 overexpression as a new strategy for induction of Golgi-specific LC3 lipidation, and the application of Golgi-damaging reagents, we unravel the mechanism and role of Golgi-LC3 lipidation. Upon DLK1 overexpression, LC3 is lipidated on the Golgi apparatus in an ATG12-ATG5-ATG16L1 complex-dependent manner; a post-Golgi trafficking blockade is the primary cause of this lipidation. During Golgi stress, ATG16L1 is recruited through its interaction with V-ATPase for Golgi-LC3 lipidation. After post-Golgi trafficking inhibition, TFE3, a key regulator of the Golgi stress response, is translocated to the nucleus. Defects in LC3 lipidation disrupt this translocation, leading to an attenuation of the Golgi stress response. Together, our results reveal the mechanism and unexplored function of Golgi-LC3 lipidation in the Golgi stress response.

TECPR1 is activated by damage-induced sphingomyelin exposure to mediate noncanonical autophagy.

Cells use noncanonical autophagy, also called conjugation of ATG8 to single membranes (CASM), to label damaged intracellular compartments with ubiquitin-like ATG8 family proteins in order to signal danger caused by pathogens or toxic compounds. CASM relies on E3 complexes to sense membrane damage, but so far, only the mechanism to activate ATG16L1-containing E3 complexes, associated with proton gradient loss, has been described. Here, we show that TECPR1-containing E3 complexes are key mediators of CASM in cells treated with a variety of pharmacological drugs, including clinically relevant nanoparticles, transfection reagents, antihistamines, lysosomotropic compounds, and detergents. Interestingly, TECPR1 retains E3 activity when ATG16L1 CASM activity is obstructed by the Salmonella Typhimurium pathogenicity factor SopF. Mechanistically, TECPR1 is recruited by damage-induced sphingomyelin (SM) exposure using two DysF domains, resulting in its activation and ATG8 lipidation. In vitro assays using purified human TECPR1-ATG5-ATG12 complex show direct activation of its E3 activity by SM, whereas SM has no effect on ATG16L1-ATG5-ATG12. We conclude that TECPR1 is a key activator of CASM downstream of SM exposure.

Conjugation of ATG8s to single membranes at a glance.

Autophagy refers to a set of degradative mechanisms whereby cytoplasmic contents are targeted to the lysosome. This is best described for macroautophagy, where a double-membrane compartment (autophagosome) is generated to engulf cytoplasmic contents. Autophagosomes are decorated with ubiquitin-like ATG8 molecules (ATG8s), which are recruited through covalent lipidation, catalysed by the E3-ligase-like ATG16L1 complex. LC3 proteins are ATG8 family members that are often used as a marker for autophagosomes. In contrast to canonical macroautophagy, conjugation of ATG8s to single membranes (CASM) describes a group of non-canonical autophagy processes in which ATG8s are targeted to pre-existing single-membrane compartments. CASM occurs in response to disrupted intracellular pH gradients, when the V-ATPase proton pump recruits ATG16L1 in a process called V-ATPase-ATG16L1-induced LC3 lipidation (VAIL). Recent work has demonstrated a parallel, alternative axis for CASM induction, triggered when the membrane recruitment factor TECPR1 recognises sphingomyelin exposed on the cytosolic face of a membrane and forms an alternative E3-ligase-like complex. This sphingomyelin-TECPR1-induced LC3 lipidation (STIL) is independent of the V-ATPase and ATG16L1. In light of these discoveries, this Cell Science at a Glance article summarises these two mechanisms of CASM to highlight how they differ from canonical macroautophagy, and from each other.

LC3B labeling of the parasitophorous vacuole membrane of Plasmodium berghei liver stage parasites depends on the V-ATPase and ATG16L1.

The protozoan parasite Plasmodium, the causative agent of malaria, undergoes an obligatory stage of intra-hepatic development before initiating a blood-stage infection. Productive invasion of hepatocytes involves the formation of a parasitophorous vacuole (PV) generated by the invagination of the host cell plasma membrane. Surrounded by the PV membrane (PVM), the parasite undergoes extensive replication. During intracellular development in the hepatocyte, the parasites provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterized by a long-lasting association of the autophagy marker protein, and ATG8 family member, LC3B with the PVM. LC3B localization at the PVM does not follow the canonical autophagy pathway since upstream events specific to canonical autophagy are dispensable. Here, we describe that LC3B localization at the PVM of Plasmodium parasites requires the V-ATPase and its interaction with ATG16L1. The WD40 domain of ATG16L1 is crucial for its recruitment to the PVM. Thus, we provide new mechanistic insight into the previously described PAAR response targeting Plasmodium liver stage parasites.

Absent epiglottic inversion as seen on flexible endoscopic evaluations of swallowing (FEES) is associated with a gestalt reduction in swallowing mechanics.

PURPOSE: Epiglottic inversion, which provides one layer of the requisite protection of the airway during swallowing, is dependent on a number of biomechanical forces. The aim of this study was to examine the association between swallowing mechanics, as visualized during a Modified Barium Swallow (MBS) exam, and the rating of epiglottic inversion as seen on Flexible Endoscopic Evaluation of Swallowing (FEES). METHODS: This study analyzed twenty-five adult outpatients referred for a simultaneous FEES/MBS exams. Each participant swallowed a 5 mL thin liquid bolus, which was the bolus size analyzed for this study's question. Epiglottic inversion, as seen on FEES, was rated by three independent raters. Additionally, twelve swallowing landmarks tracked the shape change of each participant's swallow on the MBS video using a MatLab-specific tracking tool. Analyses were run to determine mean differences in swallowing shape change between the swallows across 3 groups: complete, reduced, and absent epiglottic inversion, as seen on FEES. Using a Computerized Analysis of Swallowing Mechanics (CASM), canonical variate analyses and discriminant function testing were carried out. Other swallowing mechanics were also analyzed for kinematic movements to isolate the function of the hyoid and larynx. A two-sample t-test was conducted to compare mean hyolaryngeal movement between complete and incomplete epiglottic inversion groups. RESULTS: Overall swallowing shape changes were statistically significantly different between the absent, reduced, and complete epiglottic inversion groups on FEES. Canonical variate analyses revealed a significant overall effect of shape change between the groups (eigenvalue = 2.46, p < 0.0001). However, no statistically significant differences were found on hyoid excursion (p = 0.37) and laryngeal elevation (p = 0.06) kinematic measurements between patients with complete and incomplete epiglottic inversion on FEES. CONCLUSION: Epiglottic inversion on FEES is a valuable rating that infers reduced range of motion of structures that cannot be seen on FEES. This small sample of patients suggests that FEES ratings of absent epiglottic inversion may represent gestalt reduction in swallowing mechanics.

Combined antibiotic, steroid, and moisturizer for atopic dermatitis: A two-year case series of patient-reported outcomes.

BACKGROUND: Eczema treatments that target Staphylococcus aureus include topical mixtures of antimicrobial agent and corticosteroid diluted in a moisturizer base, previously described in the literature as compounded antibacterial, steroid, and moisturizer (CASM). There have been no placebo-controlled blinded studies of CASM. Thus, patient-reported outcome data may prove valuable. OBJECTIVE: To determine the patient-reported clinical course of eczema treatment with CASM. METHODS: Longitudinal case series between May 2016 and August 2018 of CASM patients/parents. Patients completed surveys including the POEM measure at start of treatment, 1 week, 2 weeks, 1 month, 3 months, 6 months, 9 months, and 12 months thereafter. Response curves were fitted to severity measures over time and compared by patient age group; survival analysis was used to estimate time-to-clear status as a function of patient age and initial severity. RESULTS: A total of 2153 reports were received from 728 unique respondents with a median patient age of 7 and range of 0-85. Response curves showed significant improvement over time leading to plateaus between 30 and 60 days of treatment. Overall mean improvement between start and day 90 for POEM was 14.3 (from 20.0 [95% CI 11.4-28.8] to 5.7 [95% CI -3.2 to 14.8]). Improvement was seen in each age group. CONCLUSIONS: Patient-reported outcomes suggest efficacy of CASM. There were large reductions in POEM scores, mostly in the first 30 days of treatment for all age groups.

The separate axes of TECPR1 and ATG16L1 in CASM.

Conjugation of ATG8 to single membranes (CASM) is a fundamental cellular process that entails the conjugation of mammalian Atg8 homologs, here referred to as ATG8, to phosphatidylethanolamine (PE) and phosphatidylserine (PS) on endolysosomal compartments. Our current research, together with recent reports from the Randow, Wu, and Wileman labs, has uncovered yet another layer to this process. We discovered that, in addition to ATG16L1-containing complexes, TECPR1 (tectonin beta-propeller repeat containing 1)-containing ATG12-ATG5 E3 complexes can facilitate CASM, thereby providing a broader understanding of this pathway.

Individual Atg8 paralogs exhibit unique properties in streptococcus pneumoniae-induced hierarchical autophagy.

Individual Atg8 (autophagy related 8) paralogs, comprising MAP1LC3A/LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2/GATE16, play a crucial role in canonical macroautophagy/autophagy. However, their functions remain unclear owing to functional redundancy. In a previous study, we reported that intracellular Streptococcus pneumoniae triggers hierarchical autophagy in response to bacterial infection. This process commences with the induction of conjugation of Atg8 paralogs (Atg8s) to single membranes (CASM), followed by CASM shedding and subsequent induction of xenophagy. In our recent study, we performed functional analysis of Atg8s during pneumococci-induced hierarchical autophagy. Our findings suggest that LC3A and GABARAPL1 are crucial for CASM induction, whereas GABARAPL2 and GABARAP play sequential roles in CASM shedding and subsequent induction of xenophagy, respectively.Abbreviation: Atg8: autophagy related 8; Atg8s: Atg8 paralogs; CASM: conjugation of Atg8s to single membranes; mpi: minutes post-infection; mpi: minutes post-infection; PcAV: pneumococci-containing autophagic vesicles; PcLV: LC3-associated phagosome (LAPosome)-like vacuole; PcV: pneumococci-containing vesicles; Sp: S. pneumoniae.

Membrane atg8ylation in Canonical and Noncanonical Autophagy.

Membrane atg8ylation is a homeostatic process responding to membrane remodeling and stress signals. Membranes are atg8ylated by mammalian ATG8 ubiquitin-like proteins through a ubiquitylation-like cascade. A model has recently been put forward which posits that atg8ylation of membranes is conceptually equivalent to ubiquitylation of proteins. Like ubiquitylation, membrane atg8ylation involves E1, E2 and E3 enzymes. The E3 ligases catalyze the final step of atg8ylation of aminophospholipids in membranes. Until recently, the only known E3 ligase for membrane atg8ylation was ATG16L1 in a noncovalent complex with the ATG12-ATG5 conjugate. ATG16L1 was first identified as a factor in canonical autophagy. During canonical autophagy, the ATG16L1-based E3 ligase complex includes WIPI2, which in turn recognizes phosphatidylinositiol 3-phosphate and directs atg8ylation of autophagic phagophores. As an alternative to WIPIs, binding of ATG16L1 to the proton pump V-ATPase guides atg8ylation of endolysosomal and phagosomal membranes in response to lumenal pH changes. Recently, a new E3 complex containing TECPR1 instead of ATG16L1, has been identified that responds to sphingomyelin's presence on the cytofacial side of perturbed endolysosomal membranes. In present review, we cover the principles of membrane atg8ylation, catalog its various presentations, and provide a perspective on the growing repertoire of E3 ligase complexes directing membrane atg8ylation at diverse locations.

CASM mediates LRRK2 recruitment and activation under lysosomal stress.

Conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments has attracted attention as the non-autophagic function of the Atg8-family protein conjugation system, and the V-ATPase-ATG16L1 axis has emerged as a core mechanism. Our recent research has revealed that this mechanism contributes to the lysosomal recruitment and activation of LRRK2, a Parkinson disease-associated kinase that phosphorylates a subset of RAB GTPases. The activated LRRK2 under CASM-causing lysosomal stress acts to regulate lysosomal morphology and stimulate extracellular secretion of lysosomal contents, thereby promoting the lysosomal stress response.

Individual Atg8 paralogs and a bacterial metabolite sequentially promote hierarchical CASM-xenophagy induction and transition.

Atg8 paralogs, consisting of LC3A/B/C and GBRP/GBRPL1/GATE16, function in canonical autophagy; however, their function is controversial because of functional redundancy. In innate immunity, xenophagy and non-canonical single membranous autophagy called "conjugation of Atg8s to single membranes" (CASM) eliminate bacteria in various cells. Previously, we reported that intracellular Streptococcus pneumoniae can induce unique hierarchical autophagy comprised of CASM induction, shedding, and subsequent xenophagy. However, the molecular mechanisms underlying these processes and the biological significance of transient CASM induction remain unknown. Herein, we profile the relationship between Atg8s, autophagy receptors, poly-ubiquitin, and Atg4 paralogs during pneumococcal infection to understand the driving principles of hierarchical autophagy and find that GATE16 and GBRP sequentially play a pivotal role in CASM shedding and subsequent xenophagy induction, respectively, and LC3A and GBRPL1 are involved in CASM/xenophagy induction. Moreover, we reveal ingenious bacterial tactics to gain intracellular survival niches by manipulating CASM-xenophagy progression by generating intracellular pneumococci-derived H2O2.

CASM-AMFMNet: A Network Based on Coordinate Attention Shuffle Mechanism and Asymmetric Multi-Scale Fusion Module for Classification of Grape Leaf Diseases.

Grape disease is a significant contributory factor to the decline in grape yield, typically affecting the leaves first. Efficient identification of grape leaf diseases remains a critical unmet need. To mitigate background interference in grape leaf feature extraction and improve the ability to extract small disease spots, by combining the characteristic features of grape leaf diseases, we developed a novel method for disease recognition and classification in this study. First, Gaussian filters Sobel smooth de-noising Laplace operator (GSSL) was employed to reduce image noise and enhance the texture of grape leaves. A novel network designated coordinated attention shuffle mechanism-asymmetric multi-scale fusion module net (CASM-AMFMNet) was subsequently applied for grape leaf disease identification. CoAtNet was employed as the network backbone to improve model learning and generalization capabilities, which alleviated the problem of gradient explosion to a certain extent. The CASM-AMFMNet was further utilized to capture and target grape leaf disease areas, therefore reducing background interference. Finally, Asymmetric multi-scale fusion module (AMFM) was employed to extract multi-scale features from small disease spots on grape leaves for accurate identification of small target diseases. The experimental results based on our self-made grape leaf image dataset showed that, compared to existing methods, CASM-AMFMNet achieved an accuracy of 95.95%, F1 score of 95.78%, and mAP of 90.27%. Overall, the model and methods proposed in this report could successfully identify different diseases of grape leaves and provide a feasible scheme for deep learning to correctly recognize grape diseases during agricultural production that may be used as a reference for other crops diseases.

The V-ATPase complex regulates non-canonical Atg8-family protein lipidation through ATG16L1 recruitment.

Conjugation of the Atg8 (autophagy related 8) family of ubiquitin-like proteins to phospholipids of the phagophore is a hallmark of macroautophagy/autophagy. Consequently, Atg8 family members, especially LC3B, are commonly used as a marker of autophagosomes. However, the Atg8 family of proteins are not found solely attached to double-membrane autophagosomes. In non-canonical Atg8-family protein lipidation they become conjugated to single membranes. We have shown that this process is triggered by recruitment of ATG16L1 by the vacuolar-type H+-translocating ATPase (V-ATPase) proton pump, suggesting a role for pH sensing in recruitment of Atg8-family proteins to single membranes.

Autophagy, Acute Pancreatitis and the Metamorphoses of a Trypsinogen-Activating Organelle.

Recent studies have highlighted the importance of autophagy and particularly non-canonical autophagy in the development and progression of acute pancreatitis (a frequent disease with considerable morbidity and significant mortality). An important early event in the development of acute pancreatitis is the intrapancreatic activation of trypsinogen, (i.e., formation of trypsin) leading to the autodigestion of the organ. Another prominent phenomenon associated with the initiation of this disease is vacuolisation and specifically the formation of giant endocytic vacuoles in pancreatic acinar cells. These organelles develop in acinar cells exposed to several inducers of acute pancreatitis (including taurolithocholic acid and high concentrations of secretagogues cholecystokinin and acetylcholine). Notably, early trypsinogen activation occurs in the endocytic vacuoles. These trypsinogen-activating organelles undergo activation, long-distance trafficking, and non-canonical autophagy. In this review, we will discuss the role of autophagy in acute pancreatitis and particularly focus on the recently discovered LAP-like non-canonical autophagy (LNCA) of endocytic vacuoles.

A comparative study of the modeled effects of atrazine on aquatic plant communities in midwestern streams.

Potential effects of atrazine on the nontarget aquatic plants characteristic of lower-order streams in the Midwestern United States were previously assessed using the Comprehensive Aquatic System Model (CASMATZ ). Another similar bioenergetics-based, mechanistic model, AQUATOX, was examined in the present study, with 3 objectives: 1) to develop an AQUATOX model simulation similar to the CASMATZ model reference simulation in describing temporal patterns of biomass production by modeled plant populations, 2) to examine the implications of the different approaches used by the models in deriving plant community-based levels of concern (LOCs) for atrazine, and 3) to determine the feasibility of implementing alternative ecological models to assess ecological impacts of atrazine on lower-order Midwestern streams. The results of the present comparative modeling study demonstrated that a similar reference simulation to that from the CASMATZ model could be developed using the AQUATOX model. It was also determined that development of LOCs and identification of streams with exposures in excess of the LOCs were feasible with the AQUATOX model. Compared with the CASMATZ model results, however, the AQUATOX model consistently produced higher estimates of LOCs and generated non-monotonic variations of atrazine effects with increasing exposures. The results of the present study suggest an opportunity for harmonizing the treatments of toxicity and toxicity parameter estimation in the CASMATZ and the AQUATOX models. Both models appear useful in characterizing the potential impacts of atrazine on nontarget aquatic plant populations in lower-order Midwestern streams. The present model comparison also suggests that, with appropriate parameterization, these process-based models can be used to assess the potential effects of other xenobiotics on stream ecosystems.

Propiedades psicométricas del Inventario de Hábitos de Estudio CASM-85: un estudio multicéntrico con estudiantes de secundaria peruanos / Psychometric properties of the Inventory of Study Habits CASM-85: a multicenter study with Peruvian high school students

Objetivo: evaluar la estructura interna, invarianza de medida, validez de criterio y confiabilidad del CASM-85 en estudiantes de secundaria peruanos. Materiales y Métodos: se evaluaron 2075 estudiantes de ambos sexos, entre 11 y 18 años, provenientes de colegios nacionales y particulares de Arequipa, Chimbote, Lima y Pucallpa. Se realizó un análisis de la correlación ítem-ítem e ítemresto para cada dimensión del CASM-85, un análisis factorial confirmatorio y modelos MIMIC (Multiple Indicator, Multiple Cause) para evaluar la invarianza de medida a través de grupos según sexo, edad, colegio y ciudad. La confiabilidad fue estimada mediante los coeficientes Alpha y Omega. La validez de criterio entre el CASM-85 y el rendimiento académico se revisó a través de la r de Pearson y modelos de regresión. Resultados: se eliminaron ítems heterogéneos y se obtuvo una forma reducida de 45 ítems a compararse con su original de 53 ítems. Para la forma original el CFI y TLI fue < .9 en todas las ciudades, mientras que para la forma reducida fue < . 9 solo en Chimbote. Los SRMR y RMSEA fueron ≤ .10 y ≤ .09 para las formas original y reducida respectivamente. Los modelos MIMIC confirmaron invarianza para todas las variables excepto para la variable ciudad con la forma original (│ΔCFI│ = .09, │ΔTLI│ = .08). La confiabilidad fue moderada tanto para la forma original (Ω ≥ .68; α ≥ .81) como para la reducida (Ω ≥ .77; α ≥ .84). La r de Pearson mostró una relación positiva y de fuerza débil para la forma original (r = .17) y reducida (r = .18). Conclusión: la forma reducida del CASM-85 (45 ítems) ha mostrado mejores propiedades psicométricas que su forma original.

Objective: To assess the internal structure, measurement invariance, criterion validity and reliability of the CASM-85 in Peruvian high school students. Materials and methods: Two thousand seventy-five (2075) students of both sexes, aged 11 to 18 years, from public and private high schools of Arequipa, Chimbote, Lima and Pucallpa were evaluated. An item-item and item-rest correlation analysis for each dimension of the CASM- 85, a confirmatory factorial analysis and MIMIC (Multiple Indicator, Multiple Cause) models were used to evaluate the measurement invariance across the groups according to their sex, age, school and city. Reliability was estimated using alpha and omega coefficients. The criterion validity between the CASM-85 and academic performance was determined using Pearson’s r and regression models. Results: The heterogeneous items were removed, obtaining a 45-item short form compared to the original 53-item form. In the original form, CFI and TLI were < .9 in all the cities, whereas in the short form, CFI and TLI were < .9 only in Chimbote. SRMR and RMSEA were ≤ .10 and ≤ .09 in the original and short forms, respectively. The MIMIC models confirmed the measurement invariance in all variables, with the exception of the original form between cities (│ΔCFI│ = .09, │ΔTLI│= .08). Reliability was moderate for both the original (Ω ≥ .68; α ≥ .81) and short forms (Ω ≥ .77; α ≥ .84). Pearson’s r showed a positive and weak correlation in the original (r = .17, p < .001) and short (r = .18, p < .001) forms. Conclusion: The CASM-85 short form (45 items) has shown better psychometric properties than its original full form.

Modeling the potential effects of atrazine on aquatic communities in midwestern streams.

The comprehensive aquatic systems model for atrazine (CASM(ATZ)) estimates the potential toxic effects of atrazine on populations of aquatic plants and consumers in a generic lower-order midwestern stream. The CASM(ATZ) simulates the daily production of 20 periphyton and 6 aquatic vascular plant species. The modeled consumer community consists of 17 functionally defined species of zooplankton, benthic invertebrates, bacteria, and fish. Daily values of population biomass (grams of carbon per square meter) are calculated as nonlinear functions of population bioenergetics, physical-chemical environmental parameters, grazing/predator-prey interactions, and population-specific direct and indirect responses to atrazine. The CASM(ATZ) uses Monte Carlo methods to characterize the implications of phenotypic variability, environmental variability, and uncertainty associated with atrazine toxicity data in estimating the potential impacts of time-varying atrazine exposures on population biomass and community structure. Comparisons of modeled biomass values for plants and consumers with published data indicate that the generic reference simulation realistically describes ecological production in lower-order midwestern streams. Probabilistic assessments were conducted using the CASM(ATZ) to evaluate potential modeled changes in plant community structure resulting from measured atrazine exposure profiles in 3 midwestern US streams representing watersheds highly vulnerable to runoff. Deviation in the median values of maximum 30-d average Steinhaus similarity index ranged from 0.09% to 2.52% from the reference simulation. The CASM(ATZ) could therefore be used for the purposes of risk assessment by comparison of site monitoring-based model output to a biologically relevant Steinhaus similarity index level of concern. Used as a generic screening technology or in site-specific applications, the CASM(AT) provides an effective, coherent, and transparent modeling framework for assessing ecological risks posed by pesticides in lower-order streams.