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As a key regulator of development, organ size, tissue homeostasis and cancer, the Hippo pathway is tightly regulated by various growth-related signaling events. Among them, energy stress activates the Hippo pathway to inhibit its downstream effector YAP1. Our recent work reported a YAP1-independent role of the Hippo pathway in promoting macroautophagy/autophagy and cell survival in response to energy stress, a process mediated by Hippo kinase MAP4K2. MAP4K2 interacts with and phosphorylates MAP1LC3A/LC3A at S87, which in turn drives autophagosome-lysosome fusion via the RAB3GAP-RAB18 axis. Energy stress activates MAP4K2 by reducing its association with the Hippo phosphatase complex STRIPAK component STRN4. Moreover, MAP4K2 is highly expressed in head and neck cancer, while MAP4K2 and its mediated autophagy are required for head and neck cancer development. Taken together, our study not only reveals a noncanonical role of the Hippo pathway in energy stress response, but also suggests Hippo kinase MAP4K2 as a potential therapeutic target for head and neck cancer treatment.Abbreviation: AMPK: 5'-AMP-activated protein kinase; Atg8: autophagy related 8; LATS1: large tumor suppressor 1; LIR: microtubule-associated protein 1 light chain 3-interacting region; MAP1LC3A/LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP4K2: mitogen-activated protein kinase kinase kinase kinase 2; PPP2/PP2A: protein phosphatase 2; RAB3GAP: RAB3 GTPase activating protein; RAB18: RAB18, member RAS oncogene family; SLMAP: sarcolemma associated protein; STK3/MST2: serine/threonine kinase 3; STK4/MST1: serine/threonine kinase 4; STRIPAK: striatin-interacting phosphatase and kinase; STRN4: striatin, calmodulin binding protein 4; SQSTM1/p62: sequestosome 1; TEAD: TEA domain family member; ULK1: unc-51 like kinase 1; WWTR1/TAZ: WW domain containing transcription regulator 1; YAP1: yes-associated protein 1.

Background: Abdominal aortic aneurysms (AAA) are a critical global health issue with increasing prevalence. Dexmedetomidine (DEX) is a highly selective α2-adrenoceptor agonist that has previously been shown to play a protective role in AAA. Nevertheless, the mechanisms underlying its protection effect remain not fully understood. Methods: A rat AAA model was established via intra-aortic porcine pancreatic elastase perfusion with or without DEX administration. The abdominal aortic diameters of rats were measured. Hematoxylin-eosin and Elastica van Gieson staining were conducted for histopathological observation. TUNEL and immunofluorescence staining were utilized to detect cell apoptosis and α-SMA/LC3 expression in the abdominal aortas. Protein levels were determined using western blotting. Results: DEX administration repressed the dilation of aortas, alleviated pathological damage and cell apoptosis, and suppressed phenotype switching of vascular smooth muscle cells (VSMCs). Moreover, DEX activated autophagy and regulated the AMP-activated protein kinase/mammalian target of the rapamycin (AMPK/mTOR) signaling pathway in AAA rats. Administration of the AMPK inhibitor attenuated the DEX-mediated ameliorative effects on AAA in rats. Conclusion: DEX ameliorates AAA in rat models by activating autophagy via the AMPK/mTOR pathway. [ABSTRACT FROM AUTHOR]

SCAPs (Stem Cells from Apical Papilla), derived from the apex of forming wisdom teeth, extracted from teenagers for orthodontic reasons, belong to the MSCs (Mesenchymal Stromal Cells) family. They have multipotent differentiation capabilities and are a potentially powerful model for investigating strategies of clinical cell therapies. Since autophagy—a regulated self-eating process—was proposed to be essential in osteogenesis, we investigated its involvement in the SCAP model. By using a combination of chemical and genetic approaches to inhibit autophagy, we studied early and late events of osteoblastic differentiation. We showed that blocking the formation of autophagosomes with verteporfin did not induce a dramatic alteration in early osteoblastic differentiation monitored by ALP (alkaline phosphatase) activity. However, blocking the autophagy flux with bafilomycin A1 led to ALP repression. Strikingly, the mineralization process was observed with both compounds, with calcium phosphate (CaP) nodules that remained inside cells under bafilomycin A1 treatment and numerous but smaller CaP nodules after verteporfin treatment. In contrast, deletion of Atg5 or Atg7, two genes involved in the formation of autophagosomes and essential to trigger canonical autophagy, indicated that both genes could be involved differently in the mineralization process with a modification of the ALP activity while final mineralization was not altered. [ABSTRACT FROM AUTHOR]

Background/Objectives: Non-alcoholic fatty liver disease (NAFLD) is a major metabolic disorder with no established pharmacotherapy. Quercetin, a polyphenolic flavonoid, demonstrates potential hepatoprotective effects but has limited bioavailability. This study evaluates the impact of quercetin on NAFLD and assesses the roles of autophagy-related proteins in disease progression. Methods: Forty-seven male C57BL/6J mice were fed a high-fat diet (HFD) for 12 weeks to induce NAFLD, followed by quercetin treatment for 4 weeks. Mice were divided into baseline, control, and two quercetin groups, receiving low (10 mg/kg) and high (50 mg/kg) doses. Liver histology was scored using the NAFLD Activity Score (NAS). Immunohistochemistry and immunoblotting were performed to analyze autophagy markers. Results: Quercetin-treated groups showed significant reductions in NAS compared to controls (p = 0.011), mainly in steatosis and steatohepatitis. Immunohistochemistry indicated increased expression of autophagy markers LCA and p62 in quercetin groups. Western blot analysis revealed significant elevations in LC3A in the treated groups, suggesting improved autophagic activity and lipid degradation. Conclusions: Quercetin effectively reduces NAFLD severity and modulates autophagy-related proteins. These findings suggest that quercetin enhances autophagic flux, supporting its therapeutic potential for NAFLD. Additional research is needed to clarify the molecular mechanisms of quercetin and to determine the optimal dosing for clinical application. [ABSTRACT FROM AUTHOR]

Objectives: The purpose of this study is to compare the cytotoxicity of 2 bioceramic-based (Bioserra and AH Plus Bioceramic (Bio)) and 2 epoxy resin-based (Sealart and AH Plus) root canal sealers on the Saos-2 cell line. Methods: The cytotoxicity of the root canal sealers was determined using the MTT metabolic activity assay. The mRNA expression levels of Bcl-2, Beclin1, Cas-3, IL-6, LC3, MMP-9 and TNF-α genes were assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and the expression of Beclin1 and LC3 proteins were assessed using western blot (WB) method. The canal sealers’ total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) were measured using photometric kits. Results: AH Plus Bio significantly increased metabolic activity compared to the 2 resin-containing sealers, but there was no difference with Bioserra. Sealart was clearly the most cytotoxic group. QRT-PCR analysis revealed that the mRNA expressions of Bcl-2, Beclin1, Cas-3, IL-6, LC3, and MMP-9 in the Bioserra group, Bcl-2, Cas-3, and IL-6 in the Sealart group, Beclin1, Cas-3, IL-6, and MMP-9 in the AH Plus group, and Bcl2 in the AH Plus Bio group were stimulated. The OSI results showed no significant difference between the root canal sealer groups. Conclusions: In the study, the best biocompatibility results in MTT, qRT-PCR, WB and OSI were in the AH Plus Bio group. Although Bioserra, the study’s other bioceramic paste, was found to be biocompatible according to MTT and OSI measurements, it increased the expression of all genes in qRT-PCR except for TNF-α, and also increased LC3 protein levels in WB. Clinical relevance: Since periradicular tissues can be directly affected by the materials used in root canal treatment, the sealers with high biocompatibility are being developed. In vitro studies examining the biocompatibility of newly produced root canal sealers are guiding for clinical success. [ABSTRACT FROM AUTHOR]

Mast cell tumours (MCTs) are common malignant neoplasms in dogs, for which prognosis and therapeutic decisions are based on histological features and proliferation markers. Autophagy is a cellular catabolic process responsible for degrading cytoplasmic components to maintain homeostasis, alterations in which are frequently linked to tumour growth and progression. This study was conducted to investigate the occurrence of autophagy in canine MCTs and to verify its value as a prognostic indicator for dogs with the disease. Beclin-1 and LC3B expressions were investigated using immunohistochemistry, and autophagy was ultrastructurally characterised. The autophagic phenomenon was successfully visualised in neoplastic mast cells under transmission electron and immunoelectron microscopy. MCTs from dogs that died due to the disease showed higher positivity for Beclin-1 and dogs with MCTs presenting a LC3B granular immunohistochemical pattern had a significantly shorter post-surgical survival. The occurrence of autophagy is an indicator of poor prognosis. Future studies are needed to elucidate the specific mechanisms and open new opportunities to treatments targeting this cancer cell advantage.

Contactin-associated protein1 (Caspr1) plays an important role in the formation and stability of myelinated axons. In Caspr1 mutant mice, autophagy-related structures accumulate in neurons, causing axonal degeneration; however, the mechanism by which Caspr1 regulates autophagy remains unknown. To illustrate the mechanism of Caspr1 in autophagy process, we demonstrated that Caspr1 knockout in primary neurons from mice along with human cell lines, HEK-293 and HeLa, induced autophagy by downregulating the PI3K/AKT/mTOR signaling pathway to promote the conversion of microtubule-associated protein light chain 3 I (LC3-I) to LC3-II. In contrast, Caspr1 overexpression in cells contributed to the upregulation of this signaling pathway. We also demonstrated that Caspr1 knockout led to increased LC3-I protein expression in mice. In addition, Caspr1 could inhibit the expression of autophagy-related 4B cysteine peptidase (ATG4B) protein by directly binding to ATG4B in overexpressed Caspr1 cells. Intriguingly, we found an accumulation of ATG4B in the Golgi apparatuses of cells overexpressing Caspr1; therefore, we speculate that Caspr1 may restrict ATG4 secretion from the Golgi apparatus to the cytoplasm. Collectively, our results indicate that Caspr1 may regulate autophagy by modulating the PI3K/AKT/mTOR signaling pathway and the levels of ATG4 protein, both in vitro and in vivo. Thus, Caspr1 can be a potential therapeutic target in axonal damage and demyelinating diseases. [ABSTRACT FROM AUTHOR]

Atopic bronchial asthma is the most common and severe allergic disease among a wide range of similar diseases. The main pathogenesis of this disease is characterized by a disturbance of T lymphocyte homeostasis, which significantly worsens the general state of health. In atopic bronchial asthma, there is impaired process of T cell apoptosis. This entails dysregulation and maintenance of peripheral lymphocyte homeostasis. Normally, T cells must undergo apoptosis, and its products should be utilized by neighboring cells, or professional phagocytes: monocytes, macrophages, or dendritic cells. This process is altered in atopic bronchial asthma. The immune system disorders, such as autoimmunity, often result from dysregulation of lymphocyte apoptosis. This is especially true in cases of insufficient or missed clearance of apoptotic bodies. Recently, the research and medical communities pay much attention to efferocytosis, a form of phagocytosi which proceeds by removal of apoptotic cells by phagocytes by means of LC3-associated phagocytosis (LAP). This process initiates uptake of the particles due to interactions between the phagocyte plasma membrane receptors and apoptotic cell. Further on, a single-membrane phagosome is formed in the cell with the participation of certain autophagy proteins (Beclin-1, VPS34, UVRAG, ATG5, ATG12, ATG7, ATG4, ATG4, LC3). The phagosome is enriched with LC3 protein molecules and fused with lysosomes, in which the captured “cargo” is then lysed. As a part of our work, a detailed analysis of some key protein contents at the LAP pathway was carried out for peripheral blood monocytes of patients with severe bronchial asthma. It was found that the expression of Rubicon protein is increased, thus allowing to conclude that the LAP pathway is activated in monocytes of healthy donors, thus allowing phagocytosis of dying T cells. At the same time, the components characteristic of both autophagy and LC3-associated phagocytosis are activated in the monocytes of patients with severe atopic asthma. However, one should note that decreased expression of the Rubicon protein, a putative marker of LC3-associated phagocytosis, has been clearly confirmed.

Catharanthine, a component of the anticancer drug vinblastine along with vindoline, disrupts the cell cycle by interfering with mitotic spindle formation. Apart from their antioxidant properties, vinca alkaloids like catharanthine inhibit phosphodiesterase activity and elevate intracellular cAMP levels. The aim of this study was to investigate how catharantine affects apoptosis and autophagy. This study conducted experiments on HepG2 liver carcinoma cells with varying doses of catharanthine to evaluate cell death rates and viability and determine the IC50 concentration via MTT assays. The apoptotic and autophagic effects of catharanthine were assessed using flow cytometry with annexin V and PI staining, while the expression of autophagy-related genes was analyzed through quantitative PCR. Additionally, molecular docking and molecular dynamics simulations were employed to further investigate catharanthine's impact on autophagy mechanisms. The study showed that catharanthine reduced oxidative stress and triggered apoptosis in HepG2 cells in a dose-dependent manner. Catharanthine also upregulated the expression of autophagy-related genes like LC3, Beclin1, and ULK1. Notably, catharanthine increased sirtuin-1 levels, a known autophagy inducer, while decreasing Akt expression compared to untreated cells. Molecular docking results indicated rapamycin had a stronger binding affinity with FRB (−10.7 KJ/mol−1) than catharanthine (−7.3 KJ/mol−1). Additionally, molecular dynamics simulations revealed that catharanthine interacted effectively with the FRB domain of mTOR, displaying stability and a strong binding affinity, although not as potent as rapamycin. In summary, besides its cytotoxic and pro-apoptotic effects, catharanthine activates autophagy signaling pathways and induces autophagic necrosis by inhibiting mTOR. [ABSTRACT FROM AUTHOR]

Neurons are dependent on efficient quality control mechanisms to maintain cellular homeostasis and function due to their polarization and long-life span. Autophagy is a lysosomal degradative pathway that provides nutrients during starvation and recycles damaged and/or aged proteins and organelles. In neurons, autophagosomes constitutively form in distal axons and at synapses and are trafficked retrogradely to the cell soma to fuse with lysosomes for cargo degradation. How the neuronal autophagy pathway is organized and controlled remains poorly understood. Several presynaptic endocytic proteins have been shown to regulate both synaptic vesicle recycling and autophagy. Here, by combining electron, fluorescence, and live imaging microscopy with biochemical analysis, we show that the neuron-specific protein APache, a presynaptic AP-2 interactor, functions in neurons as an important player in the autophagy process, regulating the retrograde transport of autophagosomes. We found that APache colocalizes and co-traffics with autophagosomes in primary cortical neurons and that induction of autophagy by mTOR inhibition increases LC3 and APache protein levels at synaptic boutons. APache silencing causes a blockade of autophagic flux preventing the clearance of p62/SQSTM1, leading to a severe accumulation of autophagosomes and amphisomes at synaptic terminals and along neurites due to defective retrograde transport of TrkB-containing signaling amphisomes along the axons. Together, our data identify APache as a regulator of the autophagic cycle, potentially in cooperation with AP-2, and hypothesize that its dysfunctions contribute to the early synaptic impairments in neurodegenerative conditions associated with impaired autophagy. [ABSTRACT FROM AUTHOR]

Abnormal protein accumulations in the brain are linked to aging and the pathogenesis of dementia of various types, including Alzheimer's disease. These accumulations can be reduced by cell indigenous mechanisms. Among these is autophagy, whereby proteins are transferred to lysosomes for degradation. Autophagic dysfunction hampers the elimination of pathogenic protein aggregations that contribute to cell death. We had observed that the adhesion molecule L1 interacts with microtubule-associated protein 1 light-chain 3 (LC3), which is needed for autophagy substrate selection. L1 increases cell survival in an LC3-dependent manner via its extracellular LC3 interacting region (LIR). L1 also interacts with Aβ and reduces the Aβ plaque load in an AD model mouse. Based on these results, we investigated whether L1 could contribute to autophagy of aggregated Aβ and its clearance. We here show that L1 interacts with autophagy-related protein 12 (ATG12) via its LIR domain, whereas interaction with ubiquitin-binding protein p62/SQSTM1 does not depend on LIR. Aβ, bound to L1, is carried to the autophagosome leading to Aβ elimination. Showing that the mitophagy-related L1-70 fragment is ubiquitinated, we expect that the p62/SQSTM1 pathway also contributes to Aβ elimination. We propose that enhancing L1 functions may contribute to therapy in humans. [ABSTRACT FROM AUTHOR]

Polyglutamine (polyQ)-mediated spinocerebellar ataxia (SCA), including SCA1, 2, 3, 6, 7, and 17, are caused by mutant genes with expanded CAG repeats, leading to the intracellular accumulation of aggregated proteins, the production of reactive oxygen species, and cell death. Among SCA, SCA3 is caused by a mutation in the ATXN3 (ataxin-3) gene. In a circumstance of polyQ aggregation, the autophagic pathway is induced to degrade the aggregated proteins, thereby suppressing downstream deleterious effects and promoting neuronal survival. In this study, we tested the effects of synthetic indole (NC009-1, -2, -3, -6) and coumarin (LM-022, -031) derivatives as chemical chaperones to assist mutant ATXN3-Q75 folding, as well as autophagy inducers to clear aggregated protein. Among the tested compounds, NC009-1, -2, and -6 and LM-031 interfered with Escherichia coli-derived ATXN3-Q75 aggregation in thioflavin T binding and filter trap assays. In SH-SY5Y cells expressing GFP-fused ATXN3-Q75, these compounds displayed aggregation-inhibitory and neurite growth-promoting potentials compared to untreated cells. Furthermore, these compounds activated autophagy by increasing the phosphatidylethanolamine-conjugated LC3 (microtubule associated protein 1 light chain 3)-II:cytosolic LC3-I ratio in these cells. A biochemical co-immunoprecipitation assay by using a mixture of HEK 293T cell lysates containing recombinant ATXN3-Q75-Venus-C-terminus (VC) or Venus-N-terminus (VN)-LC3 protein indicated that NC009-1 and -2 and LM-031 served as an autophagosome-tethering compound (ATTEC) to interact with ATXN3-Q75 and LC3, and the interaction was further confirmed by bimolecular fluorescence complementation analysis in cells co-expressing both ATXN3-Q75-VC and VN-LC3 proteins. The study results suggest the potential of NC009-1 and -2 and LM-031 as an ATTEC in treating SCA3 and, probably, other polyQ diseases. [ABSTRACT FROM AUTHOR]

Cement and concrete are essential for the infrastructure of the modern world. The broad availability and low cost of cement production are the primary reasons for its widespread use in construction, contributing to approximately 5-8% of CO2 emissions. Using supplementary cementitious materials (SCMs) to replace part of the clinker in cement is the most successful solution to reduce carbon dioxide emissions in the worldwide cement industry. Limestone calcined clay cement (LC³) permits the substitution of cement with calcined clay and limestone. This research investigates the properties and characteristics of LC3 cement by considering the crystallinity, associated phases, and specific surface area before and after kaolin calcination, and with 50, 55, and 60% substitution of supplementary cementitious materials compared with 11 different types of kaolin clays compared to Portland cement. The results showed that LC concrete with kaolin content of 30% and 16% with 50% substitution at the age of 28 days reaches a compressive strength of about ۱,۱۹ times and an electrical resistance of ۴٫۶ and ۹,۶ times, respectively, compared to Portland cement and also in substitution up to 60%, at the age of 28 days, it reaches acceptable compressive strength and electrical resistance about ۱۸ times that of Portland cement and Portland Pozzolana cement. [ABSTRACT FROM AUTHOR]

Chlamydia are Gram-negative, obligate intracellular bacterial pathogens that infect eukaryotic cells and reside within a host-derived vacuole known as the inclusion. To facilitate intracellular replication, these bacteria must engage in host-pathogen interactions to obtain nutrients and membranes required for the growth of the inclusion, thereby sustaining prolonged bacterial colonization. Autophagy is a highly conserved process that delivers cytoplasmic substrates to the lysosome for degradation. Pathogens have developed strategies to manipulate and/or exploit autophagy to promote their replication and persistence. This review delineates recent advances in elucidating the interplay between Chlamydia trachomatis infection and autophagy in recent years, emphasizing the intricate strategies employed by both the Chlamydia pathogens and host cells. Gaining a deeper understanding of these interactions could unveil novel strategies for the prevention and treatment of Chlamydia infection. [ABSTRACT FROM AUTHOR]

Enteroviruses are a genus of small RNA viruses that are responsible for approximately one billion global infections annually. These infections range in severity from the common cold and flu-like symptoms to more severe diseases, such as viral myocarditis, pancreatitis, and neurological disorders, that continue to pose a global health challenge with limited therapeutic strategies currently available. In the current study, we sought to understand the interaction between coxsackievirus B3 (CVB3), which is a model enterovirus, and macrophage cells, as there is limited understanding of how this virus interacts with macrophage innate immune cells. Our study demonstrated that CVB3 can robustly activate macrophages without apparent viral replication in these cells. We also showed that myeloid cells lacked the viral entry receptor coxsackievirus and adenovirus receptor (CAR). However, the expression of exogenous CAR in RAW264.7 macrophages was unable to overcome the viral replication deficit. Interestingly, the CAR expression was associated with altered inflammatory responses during prolonged infection. Additionally, we identified the autophagy protein LC3 as a novel stimulus for macrophage activation. These findings provide new insights into the mechanisms of CVB3-induced macrophage activation and its implications for viral pathogenesis. [ABSTRACT FROM AUTHOR]

The Hippo pathway is known for its crucial involvement in development, regeneration, organ size control, and cancer. While energy stress is known to activate the Hippo pathway and inhibit its effector YAP, the precise role of the Hippo pathway in energy stress response remains unclear. Here, we report a YAP-independent function of the Hippo pathway in facilitating autophagy and cell survival in response to energy stress, a process mediated by its upstream components MAP4K2 and STRIPAK. Mechanistically, energy stress disrupts the MAP4K2-STRIPAK association, leading to the activation of MAP4K2. Subsequently, MAP4K2 phosphorylates ATG8-family member LC3, thereby facilitating autophagic flux. MAP4K2 is highly expressed in head and neck cancer, and its mediated autophagy is required for head and neck tumor growth in mice. Altogether, our study unveils a noncanonical role of the Hippo pathway in energy stress response, shedding light on this key growth-related pathway in tissue homeostasis and cancer.

Hybrid externally-bonded reinforcements are considered a viable technique for strengthening existing concrete structures. They combine high-performance impregnated textiles with matrices containing dispersed microfibres to foster the ductility and toughness of the composite system. In this paper, the mechanical performance of textile-reinforced strain-hardening cement-based composites (TR-SHCC) is investigated in detail. A novel high-performance inorganic binder based on limestone calcined clay cement (LC3) is reinforced with both polyethylene (PE) dispersed microfibres and carbon fibre (CF) textiles as continuous biaxial reinforcement. The CF yarns are impregnated by an automated process to improve the monolithic response under uniaxial tensile loading and to ensure high production consistency. Fully inorganic suspensions, i.e., geopolymer and cement-based, are being investigated, as they can provide superior thermal stability compared to traditional polymeric impregnating agents. Interphase adhesion is investigated by single-yarn pull-out tests, microscopy and µCT at various micro scales. On the one hand, the improved adhesion promoted by cement impregnation resulted in the finest and most diffuse crack pattern. Conversely, the strength of the overall composite is mainly governed by the tensile failure of the yarns, irrespective of the bond, and dispersed fibres consistently improve the post-cracking stage and the strength of the hybrid composites.

Autophagy is important for many physiological processes; and disordered autophagy can contribute to the pathogenesis of a broad range of systemic disorders. C. elegans is a useful model organism for studying the genetics of autophagy, however, current methods for studying autophagy are labor-intensive and not readily amenable to high-throughput procedures. Here we describe a fluorescent reporter, GFP::LGG-1::mKate2, which is useful for monitoring autophagic flux in live animals. In the intestine, the fusion protein is processed by endogenous ATG-4 to generate GFP::LGG-1 and mKate2 proteins. We provide data indicating that the GFP:mKate ratio is a suitable readout for measuring cellular autophagic flux. Using this reporter, we measured autophagic flux in L1 larvae to day 7 adult animals. We show that basal autophagic flux is relatively low during larval development but increases markedly in reproductive adults before decreasing with age. Furthermore, we show that wild-type, eat-2, and daf-2 mutant animals have distinct autophagic flux profiles through post-embryonic development. Finally, we demonstrate the utility of this reporter by performing a high-content small molecule screen to identify compounds that alter autophagic flux in C. elegans.

Much is still unknown about microautophagy and its regulators. In our recent paper, one such regulator of microautophagy, the lipid kinase PIKfyve, is described. Previously it was found that treating cells with agents like lysomotropic drugs or proton ionophores, which alter lysosomal osmotic potential and pH, leads to a form of microautophagy that selectively degrades transmembrane proteins. Induction of this type of microautophagy is linked to a lysosomal stress response that involves the targeting of macroautophagy proteins, like ATG8s, to the lysosome membrane, through a mechanism called CASM. We found that CASM-induced microautophagy turns over ATG8s and other lysosomal membrane proteins, and requires PIKfyve activity functioning downstream of ATG8 lipidation. The lysosome biogenesis transcription factor TFEB is induced in parallel to microautophagy, in a CASM-dependent, but PIKfyve-independent manner. These findings demonstrate that stressors that engage CASM cause selective turnover by microautophagy that is coordinated with lysosome biogenesis through a mechanism that is separable through PIKfyve.

Postischemic neuroinflammation is a critical pathophysiological process within the entire pattern of cerebral ischemia. It is characterized by microglial and astroglial activation and is accompanied by disturbances in the innate and adaptive immune response. The early damage of the blood-brain barrier (BBB) integrity is accompanied by the brain autoantigens release into circulation, in particular, the neurospecific protein S100B. According to recent experimental data, activated autophagy is associated with postischemic neuroinflammation, involved in its regulation and influences the outcome of the ischemic stroke (IS) acute period. Experimental evidence is provided for the autophagy involvement in the regulation of proinflammatory cytokines and chemokines production. The influence of activated autophagy on the pro- and anti-inflammatory cytokines balance in acute IS has been demonstrated. Purpose of the study: to quantitatively evaluate key autophagy biomarkers, the early biomarker of BBB damage S100B, pro- and anti-inflammatory cytokines in the dynamics of the IS acute period. To identify the relationship between autophagy and inflammation biomarkers, 112 patients with acute IS and 56 healthy persons were examined. Patients underwent dynamic clinical neurological examination and blood testing on the 1st, 7th and 14th days from the disease’s onset. The level of autophagy in peripheral blood leukocytes was determined by flow cytometry by assessing the intracellular expression of autophagy proteins LC3, p62 and mean fluorescence intensity of the Cyto-ID dye, which specifically recognizes active autophagosomes. Serum concentrations of TNFα, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-18, neuropeptide S100B and autophagy biomarkers Beclin-1, LC3, p62 were determined by ELISA. A statistically significant increase in the studied biomarkers was found compared to the control group. The maximum increase in inflammation indicators and neuropeptide S100B was observed on the 1st, and autophagy biomarkers – on the 7th day of the disease. Established correlations indicate the participation of activated autophagy in the postischemic neuroinflammation regulation and its involvement in ischemic brain damage in the early stages of the IS acute period (days 1-7).

Background and aims: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. In this study, we aimed to investigate the role and regulatory mechanism of Annexin A2 (ANXA2) in the pathogenesis of NAFLD. Methods: Histological analyses and ELISA were used to illuminate the expression of ANXA2 in NAFLD and healthy subjects. The role of ANXA2 was evaluated using high-fat diet (HFD)-fed mice via vein injection of adeno-associated viruses (AAV) knocking down ANXA2 or non-targeting control (NC) shRNAs. Moreover, HepG2 and LO2 cells were employed as in vitro hepatocyte models to investigate the expression and function of ANXA2. Results: ANXA2 was confirmed to be one of three hub genes in liver injury, and its expression was positively correlated with NAFLD activity score (NAS) and macrophage infiltration in NAFLD. Moreover, ANXA2 was significantly upregulated in NAFLD patients and HFD-fed mice. LPS/TLR4 pathway strongly upregulated ANXA2 expression, which is mediated by direct ANXA2 promoter binding by TLR4 downstream NF-κB p65 and c-Jun transcription factors. Increased ANXA2 expression was correlated with decreased autophagy flux and autophagy was activated by the depletion of ANXA2 in the models of NAFLD. Furthermore, ANXA2 interference led to the activation of AMPK/mTOR signaling axis, which may play a causal role in autophagy flux and the amelioration of steatosis. Conclusions: ANXA2 is a pathological predictor and promising therapeutic target for NAFLD. ANXA2 plays a crucial role in linking inflammation to hepatic metabolic disorder and injury, mainly through the blockage of AMPK/mTOR-mediated lipophagy. [ABSTRACT FROM AUTHOR]

Autophagy is a highly conserved catabolic mechanism by which unnecessary or dysfunctional cellular components are removed. The dysregulation of autophagy has been implicated in various neurodegenerative diseases, including Alzheimer's disease (AD). Understanding the molecular mechanism(s)/molecules that influence autophagy may provide important insights into developing therapeutic strategies against AD and other neurodegenerative disorders. Engulfment adaptor phosphotyrosine-binding domain-containing protein 1 (GULP1) is an adaptor that interacts with amyloid precursor protein (APP) to promote amyloid-β peptide production via an unidentified mechanism. Emerging evidence suggests that GULP1 has a role in autophagy. Here, we show that GULP1 is involved in autophagy through an interaction with autophagy-related 14 (ATG14), which is a regulator of autophagosome formation. GULP1 potentiated the stimulatory effect of ATG14 on autophagy by modulating class III phosphatidylinositol 3-kinase complex 1 (PI3KC3-C1) activity. The effect of GULP1 is attenuated by a GULP1 mutation (GULP1m) that disrupts the GULP1–ATG14 interaction. Conversely, PI3KC3-C1 activity is enhanced in cells expressing APP but not in those expressing an APP mutant that does not bind GULP1, which suggests a role of GULP1–APP in regulating PI3KC3-C1 activity. Notably, GULP1 facilitates the targeting of ATG14 to the endoplasmic reticulum (ER). Moreover, the levels of both ATG14 and APP are elevated in the autophagic vacuoles (AVs) of cells expressing GULP1, but not in those expressing GULP1m. APP processing is markedly enhanced in cells co-expressing GULP1 and ATG14. Hence, GULP1 alters APP processing by promoting the entry of APP into AVs. In summary, we unveil a novel role of GULP1 in enhancing the targeting of ATG14 to the ER to stimulate autophagy and, consequently, APP processing. [ABSTRACT FROM AUTHOR]

Objective To explore the mechanism of protective effect of Jianpi Yichang Powder on intestinal mucosa by observing its effect on the expression of autophagy-related molecules in colon of ulcerative colitis (UC) model rats. Methods SD rats were randomly divided into normal group, model group, Jianpi Yichang Powder group and sulfasalazine group. UC rat model was established by 2, 4, 6-trinitrobenzene sulfonic acid/ethanol solution. After the model was successfully reproduced, the rats were given drugs orally once a day for 14 days. The general condition of rats was observed and the disease activity index (DAI) score was calculated. The pathological morphology of colonic tissue in all groups was observed by hematoxylin eosin (HE) staining. Autophagy of colonic epithelial cells was examined by transmission electron microscope. The expressions of myosin-like BCL2 interacting protein (Beclin1) and microtubule-associated protein 1 light chain 3 (LC3) in colonic tissue were detected by immunofluorescence staining and Western Blot. The mRNA expressions of autophagy protein 5 (Atg5), autophagy protein 7 (Atg7) and ubiquitin-binding protein 62 (p62) were detected by real-time quantitative polymerase chain reaction (Real-time PCR). Results Compared with the normal group, the general situation of the model group was worse, and the DAI score increased significantly (P＜0.000 1). There was obvious epithelial cell damage in colonic tissue, and the number of autophagosomes decreased obviously under electron microscope. The expressions of Beclin1 and LC3-II/ LC3-I protein as well as Atg5 and Atg7 mRNA in colonic tissue decreased significantly (P＜0.000 1), while the expression of p62 mRNA increased significantly (P＜0.000 1). Compared with the model group, the general condition of rats in Jianpi Yichang Powder group had recovered significantly, and the DAI score decreased significantly (P＜0.000 1). Pathological damage of colonic tissue has been improved in different degrees, and the number of autophagosomes increased significantly under electron microscope. The expressions of Beclin1 and LC3-II/ LC3-I protein as well as Atg5 and Atg7 mRNA in colonic tissue were significantly increased (P＜0.01, P＜0.001, P＜0.000 1), while the expression of p62 mRNA was significantly decreased (P＜0.01). Conclusion Jianpi Yichang Powder can protect UC colon mucosa from injury by up-regulating the expression of autophagy-related molecules including Beclin1, LC3, Atg5 and Atg7, down-regulating the expression of p62 and enhancing autophagy. [ABSTRACT FROM AUTHOR]

Autophagy has emerged as an important process of cell metabolism. With continuous in-depth research on autophagy, TFEB has been a key transcription factor regulating autophagy levels in recent years. Studies have established that TFEB regulates autophagy and apoptosis in various diseases. However, the relationship between TFEB and the pathogenesis of endometriosis remains unclear. This study aimed to investigate the effect of TFEB on the mechanism of endometriosis progression. The results showed that TFEB and autophagy-related protein LC3 are highly expressed in ectopic endometrium of patients with endometriosis, overexpression of TFEB in cultured human endometrial stromal cells (HESCs) by lentivirus not only promoted autophagy but also inhibited apoptosis. In addition, the migration and invasion ability of HESCs were enhanced by TFEB overexpression. Furthermore, inhibiting autophagy with specific inhibitors can attenuate migration and invasion of HESCs induced by TFEB. The rat models of endometriosis show that TFEB knockdown can suppress lesion growth in vivo. Our results suggest that autophagy may be involved in the progression mechanism of endometriosis, and the mechanism of autophagy disorder in endometriosis is probably related to TFEB. TFEB may be a key molecule in promoting endometriosis. [ABSTRACT FROM AUTHOR]

Engineered cementitious composites (ECC) are a class of high‐performing fibre‐reinforced cementitious materials recognised for their increased ductility and durability compared to conventional cement‐based materials, owing to their autogenously controlled tight crack widths, even when subjected to high strains. To reduce ECC's environmental impact, this research examines the use of a low‐clinker binder − limestone‐calcined clay cement (LC3) − as an alternative to portland cement (PC), along with fly ash to further reduce the clinker proportion and the embodied CO2 of the formulations. In conventional concrete, LC3 hydrates to a denser microstructure resulting from the synergistic reaction between limestone and calcined clay. At the lower water contents typical of ECC and with the presence of fly ash, the influence of the binder composition on the microstructure is difficult to anticipate. To examine the influence of these compositional variables on microstructure, permeability and durability, the sulphate resistance of LC3‐based ECC is explored. Specifically, the ECC‐LC3 blends are designed with high clinker replacement rate of 75% by mass of binder and contain either conventional fly ash or reclaimed fly ash at 50% by mass of binder. Expansion of ECC‐LC3 samples subjected to standard sodium sulphate test conditions was measured up to 12 months and the depth of penetration of sulphates into the ECC‐LC3 of varying compositions was quantified using micro‐X‐Ray Fluorescence (microXRF) imaging and modelling. The expansion results show that the ECC‐LC3 formulations performed better than the PC samples and can provide adequate resistance to external sulphate attack, even when reclaimed fly ashes are used in place of the conventional ash. In addition, the shallow penetration of sulphate into these cementitious composites demonstrates the low diffusion coefficients values that were determined using the quantitative data from MicroXRF imaging. [ABSTRACT FROM AUTHOR]

Autophagy is a cellular process by which proteins and organelles are degraded inside the lysosome. Exercise is known to influence the regulation of autophagy in skeletal muscle. However, as gold standard techniques to assess autophagy flux in vivo are restricted to animal research, important gaps remain in our understanding of how exercise influences autophagy activity in humans. Using available datasets, we show how the gene expression profile of autophagy receptors and ATG8 family members differ between human and mouse skeletal muscle, providing a potential explanation for their differing exercise-induced autophagy responses. Furthermore, we provide a comprehensive view of autophagy regulation following exercise in humans by summarizing human transcriptomic and phosphoproteomic datasets that provide novel targets of potential relevance. These newly identified phosphorylation sites may provide an explanation as to why both endurance and resistance exercise lead to an exercise-induced reduction in LC3B-II, while possibly divergently regulating autophagy receptors, and, potentially, autophagy flux. We also provide recommendations to use ex vivo autophagy flux assays to better understand the influence of exercise, and other stimuli, on autophagy regulation in humans. This review provides a critical overview of the field and directs researchers towards novel research areas that will improve our understanding of autophagy regulation following exercise in humans. [ABSTRACT FROM AUTHOR]

During placental development, autophagy has an important role at the molecular level, especially in cases such as trophoblast cell proliferation and cell death. Abnormal placental development due to trophoblast dysfunction causes serious gynaecological diseases and various fetal malformations. In the study conducted to investigate autophagy on the 5th day of pregnancy, in the pregnant and non pregnant group uterus tissues, uterine glands LC3 and Beclin 1 (+), in the evaluation of myometrium and perimetrium, weak (+) was observed in myometrium cells in pregnancy, while (+) reaction could not be distinguished in perimetrium. In this study, it was concluded that the immunohistochemical increase in LC3 and Beclin 1 intensity in the uterus, especially in the endometrial areas in the first trimester of pregnancy compared to the control group tissues is related to the physiology of pregnancy, homeostasis in pregnancy and hormonal mechanism. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is a malignant clonal proliferative plasma cell tumor. Zinc oxide nanoparticles (ZnO NPs) are used for antibacterial and antitumor applications in the biomedical field. This study investigated the autophagy-induced effects of ZnO NPs on the MM cell line RPMI8226 and the underlying mechanism. After RPMI8226 cells were exposed to various concentrations of ZnO NPs, the cell survival rate, morphological changes, lactate dehydrogenase (LDH) levels, cell cycle arrest, and autophagic vacuoles were monitored. Moreover, we investigated the expression of Beclin 1 (Becn1), autophagy-related gene 5 (Atg5), and Atg12 at the mRNA and protein levels, as well as the level of light chain 3 (LC3). The results showed that ZnO NPs could effectively inhibit the proliferation and promote the death of RPMI8226 cells in vitro in a dose- and time-dependent manner. ZnO NPs increased LDH levels, enhanced monodansylcadaverine (MDC) fluorescence intensity, and induced cell cycle arrest at the G2/M phases in RPMI8226 cells. Moreover, ZnO NPs significantly increased the expression of Becn1, Atg5, and Atg12 at the mRNA and protein levels and stimulated the production of LC3. We further validated the results using the autophagy inhibitor 3-methyladenine (3‑MA). Overall, we observed that ZnO NPs can trigger autophagy signaling in RPMI8226 cells, which may be a potential therapeutic approach for MM. [ABSTRACT FROM AUTHOR]

Clay is a globally abundant material and can potentially be used as a cement substitute after calcination. Though kaolinitic clay exhibits notable reactivity, the potential of low-grade clay with relatively low kaolinite content remains underexplored. This study comprehensively analyzed the chemical and mineralogical composition of marine clays at various locations and depths in Singapore, assessing their feasibility for preparing limestone calcined clay cement (LC3). The hydration kinetics and microstructure development of LC3 pastes, incorporating calcined marine clay, were investigated by XRD, isothermal calorimetry, SEM, and MIP. Additionally, a mini-migration test was employed to investigate chloride ion diffusion in LC3 pastes. The results revealed that Singapore marine clays contain relatively low kaolinite content (16–20% by QXRD). Despite a 25% reduction of 28 days compressive strength compared to reference OPC, LC3 samples exhibited outstanding resistance against chloride penetration regardless of kaolinite content (

Objective(s): Diabetes is a chronic disorder that occurs as a result of impaired glucose metabolism. In hyperglycaemic states, the balance between oxidative stress and antioxidant enzymes is disrupted leading to oxidative damage and cell death. In addition, impaired autophagy leads to the storage of dysfunctional proteins and cellular organelles in the cell. Hence, the cytoprotective function of autophagy may be disrupted by high glucose conditions. Alpha-mangostin (A-MG) is an essential xanthone purified from the mangosteen fruit. The different pharmacological benefits of alpha-mangostin, including antioxidant, anti-obesity, and antidiabetic, were demonstrated. Materials and Methods: We evaluated the protective influence of A-MG on autophagic response impaired by high concentrations of glucose in human umbilical vein endothelial cells (HUVECs). The HUVECs were treated with various glucose concentrations (5-60 mM) and A-MG (1.25-10 μM) for three days. Then, HUVECs were treated with 60 mM of glucose+2.5 μM of A-MG to measure viability, ROS, and NO content. Finally, the levels of autophagic proteins including LC3, SIRT1, and beclin 1 were evaluated by western blot.Results: The results expressed that high glucose condition (60 mM) decreased viability and increased ROS and NO content in HUVECs. In addition, LC3, SIRT1, and beclin 1 protein levels declined when HUVECs were exposed to the high concentration of glucose. A-MG reversed these detrimental effects and elevated autophagic protein levels.Conclusion: Our data represent that A-MG protects HUVECs against high glucose conditions by decreasing ROS and NO generation as well as increasing the expression of autophagy proteins.

BACKGROUND: The ARF tumour suppressor plays a well-established role as a tumour suppressor, halting cell growth by both p53-dependent and independent pathways in several cellular stress response circuits. However, data collected in recent years challenged the traditional role of this protein as a tumour suppressor. Cancer cells expressing high ARF levels showed that its expression, far from being dispensable, is required to guarantee tumour cell survival. In particular, ARF can promote autophagy, a self-digestion pathway that helps cells cope with stressful growth conditions arising during both physiological and pathological processes. METHODS: We previously showed that ARF is regulated through the activation of the protein kinase C (PKC)-dependent pathway and that an ARF phospho-mimetic mutant on the threonine residue 8, ARF-T8D, sustains cell proliferation in HeLa cells. We now explored the role of ARF phosphorylation in both basal and starvation-induced autophagy by analysing autophagic flux in cells transfected with either WT and ARF phosphorylation mutants by immunoblot and immunofluorescence. RESULTS: Here, we show that endogenous ARF expression in HeLa cells is required for starvation-induced autophagy. Further, we provide evidence that the hyper-expression of ARF-T8D appears to inhibit autophagy in both HeLa and lung cancer cells H1299. This effect is due to the cells inability to elicit autophagosomes formation upon T8D expression. CONCLUSIONS: Our results lead to the hypothesis that ARF phosphorylation could be a mechanism through which the protein promotes or counteracts autophagy. Several observations underline how autophagy could serve a dual role in cancer progression, either protecting healthy cells from damage or aiding cancerous cells to survive. Our results indicate that ARF phosphorylation controls proteins ability to promote or counteract autophagy, providing evidence of the dual role played by ARF in cancer progression.

BackgroundIn recent years the functions of astrocytes have shifted from conventional supportive roles to also include active roles in altering synapses and engulfment of cellular debris. Recent studies have implicated astrocytes in both protective and pathogenic roles impacting Huntington's disease (HD) progression.ObjectiveThe goal of this study is to determine if phagocytosis of cellular debris is compromised in HD striatal astrocytes.MethodsPrimary adult astrocytes were derived from two HD mouse models; the fast-progressing R6/2 and slower progressing Q175. With the use of laser nanosurgery, a single astrocyte was lysed within an astrocyte network. The phagocytic response of astrocytes was observed with phase contrast and by fluorescence microscopy for GFP-LC3 transiently transfected cells.ResultsAstrocyte phagocytosis was significantly diminished in primary astrocytes, consistent with the progression of HD in R6/2 and Q175 mouse models. This was defined by the number of astrocytes responding via phagocytosis and by the average number of vesicles formed per cell. GFP-LC3 was found to increasingly localize to phagocytic vesicles over a 20-min imaging period, but not in HD mice, suggesting the involvement of LC3 in astrocyte phagocytosis.ConclusionWe demonstrate a progressive decrease in LC3-associated phagocytosis in HD mouse striatal astrocytes.

Background: Autophagy is a pathway for the degradation of cytoplasmic components, which plays an essential role in various cellular and physiological processes, including cell renewal and survival, and immune responses. While recent studies have shown that they can play a role in cancer treatment, the precise mechanisms of autophagy in leukemogenesis are not fully understood. We have assessed the expression levels of LC3 and BECLIN1 as two crucial autophagy mediators in patients with leukemia. Methods: This cross-sectional study was performed on bone marrow or peripheral blood samples of 61 leukemia patients (24 AML, 20 ALL, and 17 CML) and compared to 18 healthy controls. Real-time PCR was used to quantitate gene expression. SPSS statistics 16.0 and Graph Pad Prism 8.4.2 software were applied for statistical analysis. Results: While BECLIN1 expression was significantly lower in AML, ALL, and CML patients as compared to the control group (p < 0.05), LC3 showed significantly different expression only in the AML patients (P= 0.03). There was no significant correlation between the expression levels of BECLIN1 with LC3 (p> 0.05). Whilst the AML LC3high group had a significantly lower lymphocyte count (P= 0.023), the AML BECLIN1low group had a significantly higher MPV levels (P= 0.044). Furthermore, ALL LC3high group indicated a significantly lower HCT count (P= 0.017). Conclusion: Significant changes in the expression levels of BECLINI and LC3 in hematologic malignancies may indicate a possible role for autophagy in their pathogenesis. However, further studies are warranted to confirm these findings.