The identification and characterization of noncanonical functions within the autophagy pathway have unveiled intricate cellular processes, including LC3-associated phagocytosis (LAP) and LC3-associated endocytosis (LANDO). These phenomena play pivotal roles in the conjugation of ATG8 with single-membrane phagosomes and endosomes, shedding light on the dynamic interplay between autophagy and cellular homeostasis. Here, we present detailed protocols for both qualitative and quantitative assessment of LAP, including immunofluorescence, flow cytometry, and Western blotting of isolated LAPosomes. Additionally, the protocol for the evaluation of LANDO through immunofluorescent detection of receptor recycling is outlined. The methodologies presented herein serve as a practical guide for researchers seeking to unravel the intricacies of LAP and LANDO. By providing step-by-step instructions, accompanied by insights into potential challenges and optimization strategies, this chapter aims to empower investigators in the exploration of these noncanonical functions of autophagy proteins.

UNASSIGNED: Lipid Accumulation Product (LAP) is a new type of obesity index. The relationship between LAP and depression is unclear, and this cross-sectional study was conducted to explore the relationship between LAP and depression using the National Health and Nutrition Examination Survey (NHANES) database from 2005-2018.
UNASSIGNED: In our study, logistic regression analysis was used to calculate the odds ratio between depression and LAP, and subgroup analysis and sensitivity analysis were also performed to verify the robustness of the results.
UNASSIGNED: The analysis included 13,240 participants aged 20 years or older. After adjusting for multiple variables, LAP was positively associated with depression, OR 1. 50 (95% CI, 1. 05-2. 12). In subgroup analysis, LAP was significantly positively, associated with depression among male (2. 52, OR; 95% CI, 1. 39,4. 57), non-Hispanic Black (2. 55, OR; 95% CI, 1. 49,4. 36), those without diabetes (1. 67, OR; 95% CI, (1. 06,2. 61) or in the overweight (2. 09, OR; 95% CI, (1. 23,3. 54) subgroups. After inverse probability of treatment weighting (IPTW), the OR for the highest versus lowest quartile was 1. 55 (95% CI: 1. 24 - 1. 95).
UNASSIGNED: There are positive results between LAP and depression after adjusting for multiple potential variables, and prospective studies are needed to verify the results.

OBJECTIVE: Insulin resistance is closely related to kidney function decline, but which insulin resistance index could better predict rapid kidney function decline (RKFD) remains unclear. We aimed to evaluate the prospective association between six insulin resistance indexes: Chinese Visceral Adiposity Index (CVAI), Lipid Accumulation Product (LAP), Atherogenic Index of Plasma (AIP), triglyceride-glucose (TyG) index, triglyceride-glucose × Body Mass Index (TyGBMI) and triglyceride-glucose × waist circumference (TyGWC) with RKFD and further the progression to chronic kidney disease (CKD).
METHODS: Data were obtained from the China Health and Retirement Longitudinal Study. Participants with normal kidney function (eGFRcr-cys ≥60 ml/min per 1.73 m2) and ≥45 years old were included at the baseline (year 2011). The eGFR was estimated by a combination of serum creatinine and cystatin C. The primary outcome was RKFD, defined as an annualized decline in eGFRcr-cys of 5 ml/min per 1.73 m2 or more. Secondary outcome was progression to CKD under the condition of RKFD, defined as an annualized decline in eGFRcr-cys of 5 ml/min per 1.73 m2 or more combined with eGFRcr-cys <60 ml/min per 1.73 m2 at the exit visit. Logistic analysis was applied for analysis of the association between six insulin resistance indexes and RKFD or progression to CKD. We use receiver operating characteristic curves to study the predictive performance of six insulin resistance indexes. Subgroup analysis were conducted by diabetes or hypertension status of the participants.
RESULTS: A total of 3899 participants with normal kidney function were included in this study. After a 3.99 years follow-up, 191 of them ended up with RKFD. Among them, 66 participants progressed to CKD. Logistic analysis showed that per SD increase of all the six insulin resistance indexes were significantly associated with the incidence of RKFD (all P < 0.01), among which, TyGWC had the best predictive value for RKFD. There were significant association between per SD increase of CVAI, LAP, TyGBMI and TyGWC with progression to CKD (all P < 0.01), and CVAI had better predictive role than other indexes. In subgroup analysis, we found that the association between insulin resistance indexes and progression to CKD was more significant in subjects with hypertension or without diabetes. However, no significant differences were observed in the RKFD group.
CONCLUSIONS: In this study we proved six insulin resistance indexes were predictively associated with RKFD in Chinese with normal renal function over age 45. TyGWC is the best insulin resistance index for predicting RKFD. CVAI is the best index for predicting further progression to CKD.

The β-hemolytic factor streptolysin S (SLS) is an important linear azol(in)e-containing peptide (LAP) that contributes significantly to the virulence of Streptococcus pyogenes. Despite its discovery 85 years ago, SLS has evaded structural characterizing owing to its notoriously problematic physicochemical properties. Here, we report the discovery and characterization of a structurally analogous hemolytic peptide from Enterococcus caccae, termed enterolysin S (ELS). Through heterologous expression, site-directed mutagenesis, chemoselective modification, and high-resolution mass spectrometry, we found that ELS contains an intriguing contiguous octathiazole moiety. The discovery of ELS expands our knowledge of hemolytic LAPs by adding a new member to this virulence-promoting family of modified peptides.

BACKGROUND: Skin fibrosis affects the normal function of the skin. TGF-β1 is a key cytokine that affects organ fibrosis. The latency-associated peptide (LAP) is essential for TGF-β1 activation. We previously constructed and prepared truncated LAP (tLAP), and confirmed that tLAP inhibited liver fibrosis by affecting TGF-β1. SPACE peptide has both transdermal and transmembrane functions. SPACE promotes the delivery of macromolecules through the stratum corneum into the dermis. This study aimed to alleviate skin fibrosis through the delivery of tLAP by SPACE.
METHODS: The SPACE-tLAP (SE-tLAP) recombinant plasmid was constructed. SE-tLAP was purified by nickel affinity chromatography. The effects of SE-tLAP on the proliferation, migration, and expression of fibrosis-related and inflammatory factors were evaluated in TGF-β1-induced NIH-3T3 cells. F127-SE-tLAP hydrogel was constructed by using F127 as a carrier to load SE-tLAP polypeptide. The degradation, drug release, and biocompatibility of F127-SE-tLAP were evaluated. Bleomycin was used to induce skin fibrosis in mice. HE, Masson, and immunohistochemistry were used to observe the skin histological characteristics.
RESULTS: SE-tLAP inhibited the proliferation, migration, and expression of fibrosis-related and inflammatory factors in NIH-3T3 cells. F127-SE-tLAP significantly reduced ECM production, collagen deposition, and fibrotic pathological changes, thereby alleviating skin fibrosis.
CONCLUSIONS: F127-SE-tLAP could increase the transdermal delivery of LAP, reduce the production and deposition of ECM, inhibit the formation of dermal collagen fibers, and alleviate the progression of skin fibrosis. It may provide a new idea for the therapy of skin fibrosis.

UNASSIGNED: Alveolar echinococcosis (AE) is a parasitic disease caused by E. multilocularis metacestodes and it is highly prevalent in the northern hemisphere. We have previously found that vaccination with E. multilocularis-Leucine aminopeptidase (EM-LAP) could inhibit the growth and invasion of E. multilocularis in host liver, and Ubenimex, a broad-spectrum inhibitor of LAP, could also inhibit E. multilocularis invasion but had a limited effect on the growth and development of E. multilocularis.
UNASSIGNED: In this study, the therapeutic effect of Ubenimex combined with Albendazole on AE was evaluated. Mice were intraperitoneally injected with protoscoleces and imaging examination was performed at week 8 and week 16 to detect cyst change. During this period, mice were intraperitoneally injected with Ubenimex and intragastrically administered with Albendazole suspension. At last, the therapeutic effect was evaluated by morphological and pathological examination and liver function.
UNASSIGNED: The results revealed that the combined treatment could inhibit the growth and infiltration of cysts in BALB/c mice infected with E. multilocularis protoscoleces. The weight, number, invasion and fibrosis of cysts were reduced in mice treated with Ubenimex in combination with Albendazole. The same effect was achieved by the single Ubenimex treatment because of its inhibitory effect on LAP activity, but it was less effective in inhibiting the growth of cysts. The levels of ALT, AST, TBIL, DBIL, ALP, and γ-GT were reduced after the combined treatment, indicating that treatment with both Ubenimex and Albendazole could alleviate liver damage.
UNASSIGNED: This study suggests that the combined treatment with Ubenimex and Albendazole could be a potential therapeutic strategy for E. multilocularis infections.

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.

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.

BACKGROUND: Studies have shown that lipid-related indicators are associated with testosterone deficiency. However, it is difficult to determine which indicator is the most accurate predictor of testosterone deficiency. We aimed to identify the lipid-related indicators most predictive of testosterone deficiency in adults in the United States.
METHODS: This observational research was conducted on a population aged ≥ 20 years. By plotting the receiver operating characteristic curve (ROC) and obtaining the corresponding area under the curve (AUC) value, we assessed the predictive capacity of TyG, WTI, LAP, and VAI for testosterone deficiency. We compared the area under the curve (AUC) values of these measures to determine if there were any statistically significant differences. The relationship between lipid-related indices and testosterone hormones was investigated using regression modeling, eXtreme Gradient Boosting (XGBoost) modeling, and sensitivity analysis.
RESULTS: A total of 3,272 eligible participants were included in the study. Testosterone deficiency was found to exist in 20.63% of the participants. Subjects with higher lipid-related markers were more likely to have lower testosterone levels. LAP was the best predictor of testosterone deficiency in ROC analysis over other indicators (AUC = 0.7176, (95% CI: 0.6964-0.7389)).
CONCLUSIONS: LAP is the most straightforward and convenient indicator for identifying testosterone deficiency in clinical practice.

Intracellular infections as well as changes in the cell nutritional environment are main events that trigger cellular stress responses. One crucial cell response to stress conditions is autophagy. During the last 30 years, several scenarios involving autophagy induction or inhibition over the course of an intracellular invasion by pathogens have been uncovered. In this review, we will present how this knowledge was gained by studying different microorganisms. We intend to discuss how the cell, via autophagy, tries to repel these attacks with the objective of destroying the intruder, but also how some pathogens have developed strategies to subvert this. These two fates can be compared with a Tango, a dance originated in Buenos Aires, Argentina, in which the partner dancers are in close connection. One of them is the leader, embracing and involving the partner, but the follower may respond escaping from the leader. This joint dance is indeed highly synchronized and controlled, perfectly reflecting the interaction between autophagy and microorganism.