Mesenchymal stem cells (MSCs) serve as ideal candidates for a broad range of cell-based therapies. However, cell ageing caused by long-term in vitro expansion and poor survival after in vivo delivery greatly limits their success in preclinical and clinical applications. Dedifferentiation represents a potential strategy for enhancing the retention and function of MSCs in hostile environments. In this study, we evaluated the cell phenotype, proliferation, and differentiation potential, as well as the anti-oxidative stress ability of human umbilical cord-derived MSCs (hMSCs) manipulated with adipogenic priming and subsequent dedifferentiation. After an in vitro differentiation and dedifferentiation procedure, the resultant dedifferentiated hMSCs (De-hMSCs) displayed properties similar to their original counterparts, including immunophenotype and mesodermal potential. Upon re-induction, De-hMSCs exhibited a significantly higher adipogenic differentiation capability than unmanipulated hMSCs. Importantly, De-hMSCs showed a significantly enhanced ability to resist tert-butyl hydroperoxide (t-BHP) induced apoptosis compared to undifferentiated hMSCs. Mechanisms involving bcl-2 family proteins and autophagy may contribute to the demonstrated advantages of dedifferentiation-reprogrammed hMSCs. These results indicate that adipogenic dedifferentiation promotes adipogenesis and cell persistence, as well as preserves the stemness of human umbilical cord-derived MSCs that have been committed to the adipocytic lineage. As a unique stem cell population, dedifferentiated MSCs may represent an attractive and promising candidate for MSC-based therapy.

Hypoxia-inducible factors (HIFs) mediate cellular responses to low oxygen, notably enhanced fermentation that acidifies poorly perfused tissues and may eventually become more damaging than adaptive. How pH feeds back on hypoxic signaling is unclear but critical to investigate because acidosis and hypoxia are mechanistically coupled in diffusion-limited settings, such as tumors. Here, we examined the pH sensitivity of hypoxic signaling in colorectal cancer cells that can survive acidosis. HIF-1α stabilization under acidotic hypoxia was transient, declining over 48 h. Proteomic analyses identified responses that followed HIF-1α, including canonical HIF targets (e.g., CA9, PDK1), but these did not reflect a proteome-wide downregulation. Enrichment analyses suggested a role for lysosomal degradation. Indeed, HIF-1α destabilization was blocked by inactivating lysosomes, but not proteasome inhibitors. Acidotic hypoxia stimulated lysosomal activity and autophagy via mammalian target of rapamycin complex I (mTORC1), resulting in HIF-1α degradation. This response protects cells from excessive acidification by unchecked fermentation. Thus, alkaline conditions are permissive for at least some aspects of HIF-1α signaling.

Dinutuximab beta has shown limited efficacy in treating high-risk neuroblastoma (NB). Combining autophagy inhibitors with immune checkpoint inhibitors (ICIs) has proven effective in many malignancies. However, the anti-tumor effects of autophagy inhibition in conjunction with anti-GD2 immunotherapy remain unknown. In this study, dinutuximab beta induces anti-proliferation and anti-EMT activity in NB cells. Dinutuximab beta also triggers autophagy in NB cells, and inhibition of the VEGFR pathway with anlotinib amplifies dinutuximab beta-induced autophagy. In addition, dinutuximab beta induces the synthesis of the chemokine CXCL9 and the infiltration of CD8+ T cells. Mechanistically, dinutuximab beta inhibits the VEGFR/AKT/mTOR and ROS/NF-κB pathways. Furthermore, autophagy inhibition by CQ enhances CXCL9 expression and anti-tumor T cell responses of single anti-GD2 therapy in vitro and in vivo. Collectively, this study suggests autophagy inhibitors may be a promising strategy for enhancing therapeutic efficacy in NB in conjunction with anti-GD2 immunotherapy.

Oncolytic reovirus is a potential therapeutic for colorectal cancer patients with a mutant KRAS gene. Reovirus hijacks the autophagic machinery and preferentially induces apoptosis in patients with a KRAS mutation. However, reovirus on its own is not currently a viable treatment and requires enhancement with combination therapies. Carbamazepine, an FDA-approved drug in use for epilepsy, is an autophagy inducer and is used in this study in conjunction with reovirus. The dual treatment was able to reduce cancer viability in mutated KRAS cell lines and was more effective than with reovirus alone. Carbamazepine and reovirus increased autophagy-related proteins and mRNA in mutant KRAS compared to wildtype KRAS which is crucial for autophagy-induced apoptosis. Transmission electron microscopy results show increased autophagosome formation in the combination therapy, as well as a decrease in condensed chromatin. The combination therapy effectively increased the apoptosis induced by reovirus alone and is a viable treatment for patients with a mutant KRAS.

Repeated exposure to airborne terrestrial natural minerals may cause pneumoconiosis and lung cancer, among which iron sulfide is identified as an aggravating factor. In the biological system, iron-sulfur cluster is an inorganic cofactor that is evolutionarily conserved in all the living organisms. Whereas ferrous iron catalyzes the generation of hydroxyl radicals, sulfur is indispensable as a component of antioxidants, such as glutathione. Imbalanced redox homeostasis contributes to oxidative stress, causing ferroptosis, an iron-dependent regulated necrosis characterized by lipid peroxidation, resulting in various disorders. We undertook this study to understand the cellular regulatory mechanisms against major terrestrial minerals containing iron and sulfur from the viewpoint of cellular redox. We used fundamental iron sulfide minerals collected from natural sources to treat human macrophage and fibroblast cells and investigated the biological responses. Alterations in sulfane sulfur, glutathione and iron have been analyzed using either specific fluorescent probes or inductively coupled plasma mass spectrometry. Iron sulfide microparticles with high Fe/S ratio (pyrrhotite; Fe1-XS) induced more reactive sulfane species and glutathione, with less catalytic iron inside cells, whereas the mineral with low Fe/S ratio (pyrite; FeS2) exhibited the opposite effects. Notably both showed cytotoxicity, where pyrite caused ferroptosis but pyrrhotite led to non-ferroptotic disruption. Furthermore, assimilated cellular excess iron was secreted via CD63(+) exosome containing iron-loaded ferritin to the extracellular space with higher iron content in pyrrhotite. Our findings suggest that iron and sulfur work complementarily in maintaining intracellular redox homeostasis, which would be crucial to understand the associated pathology.

Tumor suppressor genes (TSGs) that regulate the stemness of lung cancer cells remain to be determined. We conducted a genome-wide CRISPR/Cas9-mediated screening and identified REPS2 as a potent TSG that negatively regulates the stemness of lung cancer cells. Its tumor suppressive function was confirmed both in vitro and in vivo. Mechanistically, P62 interacts simultaneously with both β-catenin and REPS2, leading to autophagy-lysosome-mediated degradation of β-catenin and attenuation of Wnt signaling. A β-catenin inhibitor synergizes with inhibitors for driver mutants to induce immunogenic cell death, which could be exploited for enhancing efficacy of tumor immunotherapy.

The global aging population is increasingly inflicted with Alzheimer's disease (AD), but a cure is still unavailable. Neurotrophic Factor-α1/carboxypeptidase E (NF-α1/CPE) gene therapy has been shown to prevent and reverse memory loss and pathology AD mouse models However, the mechanisms of action of NF-α1/CPE are not fully understood. We investigated if a non-enzymatic form of NF-α1/CPE-E342Q is efficient in reversing AD pathology and carried out a proteomic study to uncover the mechanisms of action of NF-α1/CPE in AD mice.

AAV-human NF-α1/CPE and a non-enzymatic form, NF-α1/CPE -E342Q were delivered into hippocampus of 3xTg-AD mice and effects on cognitive function, neurodegeneration, synaptogenesis and autophagy were investigated. A quantitative proteomic analysis of hippocampus of 3xTg-AD mice with and without AAV-NF-α1/CPE treatment was carried out.

Hippocampal delivery of AAV-NF-α1/CPE-E342Q prevented memory loss, neurodegeneration and increase in activated microglia in 3xTg-AD mice, indicating its action is independent of its enzymatic activity. Quantitative proteomic analysis of hippocampus of 3xTg-AD mice that underwent NF-α1/CPE gene therapy revealed differential expression of >2000 proteins involving many metabolic pathways. Of these, two new proteins down-regulated by NF-α1/CPE: Nexin4 (SNX4) and Trim28 which increase Aβ production and tau levels, respectively were identified. Western blot analysis verified that they were reduced in AAV-NF-α1/CPE treated 3xTg-AD mice compared to untreated mice. Our proteomic analysis indicated synaptic organization as top signaling pathway altered as a response to CPE expression. Synaptic markers PSD95 and Synapsin1 were decreased in 3xTg-AD mice and were restored with AAV-NF-α1/CPE treatment. Proteomic analysis hypothesized involvement of autophagic signaling pathway. Indeed, multiple proteins known to be markers of autophagy were down-regulated in 3xTg-AD mice, accounting for impaired autophagy. Expression of these proteins were upregulated in 3xTg-AD mice with NF-α1/CPE gene therapy, thereby reversing autophagic impairment.

This study uncovered vast actions of NF-α1/CPE in restoring expression of networks of critical proteins including those necessary for maintaining neuronal survival, synaptogenesis and autophagy, while down-regulating many proteins that promote tau and Aβ accumulation to reverse memory loss and AD pathology in 3xTg-AD mice. AAV-NF-α1/CPE gene therapy uniquely targets many metabolic levels, offering a promising holistic approach for AD treatment.

Saccharibacteria are episymbionts that require host-bacteria to grow. They are positively associated with inflammatory diseases within the human microbiome, yet their mechanisms for interacting with the human host and contributing to diseases remain unknown. This study investigated interactions between a Saccharibacterium (Nanosynbacter lyticus), its host-bacteria (Schaalia odontolytica), and oral epithelial cells. The host-bacteria induced proinflammatory cytokines in epithelial cells, while Saccharibacteria were immune silent. Remarkably, Saccharibacteria dampened cytokine responses to host-bacteria during coinfection. This effect was driven by Saccharibacteria-induced clustering of TLR2 receptors, a process likely facilitated by type IV, ultimately leading to reduced TLR2-mediated cytokine signalling. High resolution imaging showed that Saccharibacteria were endocytosed by oral epithelial cells, and colocalized with endosome markers, eventually trafficking to lysosomes. Moreover, a subset of the Saccharibacteria survive endocytosis long-term, and retains their capability to reinfect host-bacteria, highlighting a mechanism for persistence in the oral microbiome and a vital role in mammalian immune system modulation.

Chronic pancreatitis (CP), a progressive inflammatory disease characterized by pancreatic tissue destruction and fibrosis, is considered a challenging health burden due to insufficiencies of current management procedures. Autophagy impairment has emerged as a major triggering event in pancreatitis, raising interest in exploring the potential of targeting autophagy as a possible interventional strategy. This study aimed to evaluate the possible ameliorative effect of two autophagy modulators, simvastatin and eugenol, on CP-related perturbations in an arginine-induced rat model. Repeated l-arginine administration (5 g/kg divided into 2 doses with a 1 h interval, given intraperitoneally every 3rd day for a total of 10 times) provoked CP features, demonstrated by acinar damage, oxidative stress, inflammation, and fibrosis. Arginine-triggered pancreatitis was accompanied by hampered pancreatic autophagic flux, evidenced by overexpression of pancreatic p62 and LC3-Ⅱ and downregulation of pancreatic AMPK and LAMP-1 mRNA expression. Treatment with simvastatin (20 mg/kg, intraperitoneally 24 h, before each arginine dose) and eugenol (50 mg/kg/day orally for 30 days) achieved significant anti-oxidative, anti-inflammatory, and anti-fibrotic effects, and reversed the arginine-instigated autophagic blockade, with superior ameliorative effects attained by eugenol. Altogether, simvastatin and eugenol provide a promising interventional approach for CP, at least partly, by restoring the impaired autophagic flux associated with CP.

Autophagy, a dynamic intracellular degradation system, is critical for cellular renovation and maintaining equilibrium. By eliminating damaged components and recycling essential molecules, autophagy safeguards cellular integrity and function. The versatility of the autophagy process across various biological functions enable cells to adapt and maintain homeostasis under unfavourable conditions. Disruptions in autophagy can shift a cell from a healthy state to a disease state or, conversely, support a return to health. This review delves into the multifaceted role of autophagy during aging and age-related diseases such as cancer, highlighting its significance as a unifying target with promising therapeutic implications. Cancer development is a dynamic process characterized by the acquisition of diverse survival capabilities for proliferating at different stages. This progression unfolds over time, with cancer cells exploiting autophagy to overcome encountered stress conditions during tumor development. Notably, there are several common pathways that utilize the autophagy process during aging and cancer development. This highlights the importance of autophagy as a crucial therapeutic target, holding the potential to not only impede the growth of tumor but also enhance the patient's longevity. This review aims to simplify the intricate relationship between cancer and aging, with a particular focus on the role of autophagy.

Hydrogen sulfide (H2S) holds a distinct role in cell biology. Its level is intricately linked to the homeostasis of the biological environment, underscoring the significance of developing techniques capable of detecting H2S in biological systems. A single probe that offers versatility across different detection techniques opens opportunities for advancements in sensing H2S in various fields. A nitronaphthalimide derivative, NMO prepared using a simple synthetic protocol, has been studied as an electrochemical, colorimetric, and turn-on fluorescence probe for H2S. NMO displayed a detection limit of 9.95 mM and 4.36 mM in the UV-visible and colorimetric studies, respectively, whereas the fluorometric and square wave techniques confirmed lower detection limits of 98.4 μM and 1.24 mM, correspondingly. Further, the real-time imaging of HEK293T cells using NMO during stress-induced autophagy is demonstrated.

The human endogenous retrovirus type W envelope glycoprotein (ERVWE1), located at chromosome 7q21-22, has been implicated in the pathophysiology of schizophrenia. Our previous studies have shown elevated ERVWE1 expression in schizophrenia patients. Growing evidence suggests that autophagy dysfunction contributes to schizophrenia, yet the relationship between ERVWE1 and autophagy remains unclear. In this study, bioinformatics analysis of the human prefrontal cortex RNA microarray dataset (GSE53987) revealed that differentially expressed genes were predominantly enriched in autophagy-related pathways. Clinical data further demonstrated that serum levels of microtubule-associated protein 1 light chain 3β (LC3B), a key marker of macroautophagy, were significantly elevated in schizophrenia patients compared to controls, and positively correlated with ERVWE1 expression. Cellular and molecular experiments suggested that ERVWE1 promoted macroautophagy by increasing the LC3B II/I ratio, enhancing autophagosome formation, and reducing sequestosome 1 (SQSTM1) expression via upregulation of NADPH oxidase activator 1 (NOXA1). Concurrently, NOXA1 downregulated the expression of key micromitophagy-related genes, including PTEN-induced kinase 1 (PINK1), Parkin RBR E3 ubiquitin-protein ligase (Parkin), and the pyruvate dehydrogenase E1 subunit α 1 (PDHA1). As a result, ERVWE1, via NOXA1, inhibited micromitophagy by suppressing the expression of PINK1, Parkin, and PDHA1, thereby leading to impaired production of mitochondrial-derived vesicles (MDVs). Mechanistically, ERVWE1 enhanced NOXA1 transcription by upregulating upstream transcription factor 2 (USF2). In conclusion, ERVWE1 promotes macroautophagy and inhibits micromitophagy through USF2-NOXA1 axis, providing novel mechanistic insight into the role autophagy dysregulation in schizophrenia. These findings suggest that targeting autophagy pathways may offer novel therapeutic strategies for schizophrenia treatment.

The identification of alternative cell death pathways is key to developing therapies for apoptosis-resistant cancers. We investigated cell death induced by delocalized lipophilic cation (DLC) nanoaggregates in A549 lung carcinoma cells. These DLCs trigger a dynamin-dependent, nonapoptotic pathway involving cytoplasmic vesicle accumulation and mitochondrial dysfunction. Leveraging the mitochondria-targeting ability of lipophilic cations, we designed and synthesized fluorescent mitochondrion-toxic molecules with potent cytotoxicity against A549, MDA-MB-231, and MCF-7 cells. Dynamic light scattering revealed the nanoaggregate formation of the lead compound, L3, in the RPMI media. L3 inhibited metastasis and clonal expansion, induced vacuole formation post endocytosis, and impaired the mitochondrial function, disrupting ATP levels. This led to mitochondrial permeability transition pore (MPTP) opening and oxidative imbalance via glutathione perturbation. L3 demonstrated strong antitumor activity in vitro and in vivo, showing high potential for treating apoptosis-resistant cancers.

Improving treatment options for head and neck squamous cell carcinoma (HNSCC) requires a deeper understanding of the tumor microenvironment, particularly cancer-associated fibroblasts (CAFs). We previously reported that HNSCC-derived FGF2/bFGF (fibroblast growth factor 2) triggers cytokine release from CAFs via secretory autophagy. Here, using transmission electron microscopy, live-cell imaging, and immunofluorescence, we show that CAF autophagosomes transport cargo, including IL6, to the plasma membrane for secretion. Autophagy in CAFs is constitutive and independent of STAT3, MAPK1/ERK2-MAPK3/ERK1 and phosphoinositide 3-kinase (PI3K) signaling. Despite the significant role of secretory autophagy in CAFs, its molecular machinery has remained elusive. Using both a literature based, and an unbiased approach, we studied the molecular machinery involved in autophagosome trafficking in CAFs. We identified TRIM16, a protein previously reported to traffic to autophagosomes, upregulated in CAFs compared to normal oral fibroblasts. Immunohistochemistry of patient HNSCC stroma revealed co-expression of TRIM16 and LC3B, linking TRIM16 to autophagosome function. An unbiased proteomics profiling of immunoprecipitated LC3B+ vesicles in primary HNSCC CAFs revealed enrichment in trafficking proteins, focal adhesion, and mitochondrial proteins. We demonstrate that SEC22B, SNAP23, VAMP3, and STX4 colocalize with LC3B, IL6, and TRIM16 in CAFs. TRIM16 knockdown reduced autophagosomes at the plasma membrane and decreased IL6 secretion from CAFs. These findings uncover key molecular components involved in autophagy-mediated IL6 secretion in CAFs and suggest potential therapeutic targets for HNSCC.Abbreviations: ACTA2/αSMA: actin alpha 2, smooth muscle; CAF: cancer-associated fibroblasts; CM: conditioned media; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DMSO: dimethylsulfoxide; EGFP: enhanced green fluorescent protein; ELISA: enzyme-linked immunosorbent assay; ER: endoplasmic reticulum; FGF2/bFGF: fibroblast growth factor 2; FGFR: fibroblast growth factor receptor; GO: gene ontology; GORASP2/GRASP55: golgi reassembly stacking protein 2; HMGB1: high mobility group box 1; HNSCC: head and neck squamous cell carcinoma; HPV: human papillomavirus; IL6: interleukin 6; IP: immunoprecipitation; LC-MS/MS: liquid chromatography-mass spectrometry/mass spectrometry; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; NFs: normal oral fibroblasts; NSCLC: non-small cell lung cancer; PLA: proximity ligation assay; SQSTM1/p62: sequestosome 1; STAT3: signal transducer and activator of transcription 3; SNAP23: synaptosome associated protein 23; SNARE: soluble N-ethyl-maleimide-sensitive factor attachment protein receptor; STX4: syntaxin 4; TEM: transmission electron microscopy; TGFB1: transforming growth factor beta 1; TMA: tissue microarray; TRIM: tri-partite motif; VAMP: vesicle associated membrane protein; VC: vehicle control.

Iron-induced lipid peroxidation of phosphatidylethanolamine (PE) species is a key driver of ferroptosis in retinal pigment epithelial (RPE) cells, a process closely associated with age-related macular degeneration (AMD). The previous studies have demonstrated that induced retinal pigment epithelial (iRPE) cells generated by transcription factor-mediated reprogramming exhibit superior therapeutic efficacy in treating AMD. In this study, it is found that these iRPE cells are resistant to ferroptosis and further identified phosphoethanolamine/phosphocholine phosphatase 1 (PHOSPHO1) as a critical regulator underlying ferroptosis resistance. Mechanistically, PHOSPHO1 inhibits ferroptosis through two distinct mechanisms. First, it reduces PE levels in the endoplasmic reticulum, thereby limiting PE-derived lipid peroxidation. Second, it suppresses autophagy and ferritinophagy, leading to a reduction in intracellular free iron accumulation. Experiments using an in vivo rat model confirm that PHOSPHO1 effectively protects RPE cells from ferroptotic damage. These findings highlight PHOSPHO1 as a potential therapeutic target for AMD, providing insights into novel ferroptosis-based intervention strategies.

Cadmium (Cd), an environmental toxicant, accumulates in the human body and damages the male reproductive system. To investigate the molecular mechanisms underlying Cd-induced reproductive toxicity, we used GC-2spd cells and treated them with CdCl2. Additionally, we added 2-APB (an inhibitor of the IP3R) and STF-083010 (an inhibitor of IRE1) to investigate whether they could ameliorate Cd-induced reproductive toxicity. Confocal microscopy and flow cytometry confirmed that CdCl2-treated GC-2spd cells displayed imbalance of calcium homeostasis, with upregulation of the expression of the IP3R, a key pathway for endoplasmic reticulum (ER) Ca2+ release. Furthermore, the ER stress (ERS) effector protein IRE1 expression was also increased, suggesting that Cd activated ERS and the IRE1 pathway by disrupting calcium homeostasis. Previous studies have shown that ERS induces autophagy. We performed the MDC assay to detect autophagosome formation, revealing increased expression of autophagy-related proteins LC3-II/LC3-I and Beclin-1 in response to Cd treatment. In contrast, treatment with 2-APB and STF-083010 inhibited autophagy and mitigated cell death. This inhibitory effect may be due to 2-APB blocking IP3R-mediated Ca2+ release, alleviating imbalance of calcium homeostasis, while STF-083010 inhibits IRE1, restoring ER homeostasis and reducing autophagy. These findings suggest that imbalance of calcium homeostasis activates the IRE1 pathway-mediated ERS, leading to excessive autophagy and male reproductive toxicity. Conversely, the addition of 2-APB and STF-083010 reversed these effects, synergistically restoring intracellular Ca2+ homeostasis and inhibiting ERS to promote cell health. This study provides a new therapeutic strategy for Cd-induced male reproductive disorders.

The accumulation of plastic waste in the environment, breaking down into micro- and nanoplastics, poses significant threats to ecosystem and human health. Plastic particles have been detected in human blood, urine, and placental tissue, indicating widespread exposure. While their long-term health impacts remain unclear, developing brains, especially in fetuses and children, may be vulnerable, potentially resulting in behavioral changes or neurodevelopmental disorders. This study explores the effects of chronic exposure to 23-nm polystyrene nanoplastics at 10 µg/day/kg in wild-type mice across life stages, using exposure levels reflective of human reality. Maternal exposure disrupted critical developmental milestones in pups. In adulthood, exposed mice exhibited Attention-Deficit Hyperactivity Disorder (ADHD)-like traits, including hyperactivity, increased risk-taking behaviors, and impaired motor learning and executive functions. In aging mice, exposure was associated with a lower epileptic threshold, with developing seizures. These behavioral changes were linked to altered gene and synaptic protein expression associated with ADHD and epilepsy. At the cellular level, lifelong nanoplastic exposure caused lysosomal dysfunctions and increased lipofuscin accumulation, indicative of accelerated brain aging. These findings align with the growing prevalence of ADHD and epilepsy in humans, particularly children and the elderly, emphasizing the urgent need to address plastic pollution and its health implications.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a threat due to the emergence of variants with increased transmissibility and enhanced escape from immune responses. Like other coronaviruses before, SARS-CoV-2 likely emerged after its transmission from bats. The successful propagation of SARS-CoV-2 in humans might have been facilitated by usurping evolutionarily conserved cellular factors to execute crucial steps in its life cycle, such as the generation of replication organelles-membrane structures where coronaviruses assemble their replication-transcription complex. In this study, we found that RAB5, which is highly conserved across mammals, is a critical host dependency factor for the replication of the SARS-CoV-2 genome. Our results also suggest that SARS-CoV-2 uses RAB5+ membranes to build replication organelles with the aid of COPB1, a component of the COP-I complex, and that the virus protein NSP6 participates in this process. Hence, targeting NSP6 represents a promising approach to interfere with SARS-CoV-2 RNA synthesis and halt its propagation.IMPORTANCEIn this study, we sought to identify the host dependency factors that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses for the generation of replication organelles: cellular membranous structures that SARS-CoV-2 builds in order to support the replication and transcription of its genome. We uncovered that RAB5 is an important dependency factor for SARS-CoV-2 replication and the generation of replication organelles, and that the viral protein NSP6 participates in this process. Hence, NSP6 represents a promising target to halt SARS-CoV-2 replication.

Acute lung injury (ALI), including its most severe form, acute respiratory distress syndrome (ARDS), is a common cause of acute hypoxemic respiratory failure. Although its clinical characteristics have been well characterized, the relevant mechanism remains unclear. An imbalance in autophagy leads to alveolar remodeling and triggers the pathogenesis of ARDS. In this study, we assessed the therapeutic efficacy of the STAT1 inhibitor fludarabine (Fluda) in ALI. C57BL6 mice were exposed to lipopolysaccharide (LPS), and their lung tissues were analyzed via next-generation transcriptome sequencing.

Western blotting revealed that interferon regulatory factor 1 (IRF1) was highly expressed and STAT1 was phosphorylated following LPS exposure. Fluda significantly decreased the protein expression of STAT1/IRF1 and inhibited the alveolar infiltration of neutrophils and macrophages. Nitric oxide (NO), inducible nitric oxide synthase, tumor necrosis factor-α (TNF-α), interferon-γ, and interleukin-6 (IL-6) release was decreased in the lungs of mice and RAW264.7 macrophages following Fluda treatment. In LPS-induced GFP-LC3 transgenic mice treated with Fluda, the counts of LC3-expressing neutrophils and macrophages in bronchoalveolar (BAL) fluid were significantly decreased. Furthermore, Fluda decreased LC3 and p62 protein expression, thereby inhibiting the release of NO, IL-6, and TNF-α in BAL. In RAW264.7 cells, the inhibition of STAT1/IRF1 by Fluda decreased LPS-induced ERK and NF-κB p65 phosphorylation.

The inhibition of STAT1/IRF1 by Fluda plays a pivotal role in modulating dysregulated autophagy by suppressing the MAPK and NF-κB p65 pathways in ALI.

Endothelial cells respond to flow-induced shear stress by morphological changes, a process which is important for vascular development and physiology. High laminar shear stress activates Tie-2 which supports endothelial junction integrity and protects against vascular leaks and the generation of atherosclerotic plaques.

We have examined the role of Tie-2 and FOXO1 in controlling vascular endothelial cell morphology under physiological shear stress. To address this, we exposed human umbilical vein endothelial cells (HUVECs) transfected with siRNA to 15 dyn/cm2 of shear stress for 24 hours. The resulting cells were analyzed by immunofluorescence staining.

We found that shear stress-induced activation of Tie-2 is required for endothelial cell alignment and elongation in the direction of flow. Mechanistically, we found that FOXO1 is an essential target downstream of Tie-2, which becomes translocated from the nucleus into the cytosol. There, FOXO1 stimulates the formation of autophagosomes, and both FOXO1 and autophagy stimulation are needed for Tie-2-dependent cell alignment.

In conclusion, laminar fluid shear stress stimulates a novel Tie-2-FOXO1-autophagy signaling axis which is required for endothelial cell alignment. This represents a new mechanism by which Tie-2 contributes to vascular protection under laminar shear stress.

Background/Objectives: Autophagy is a conserved self-degradation process by which cells break down and recycle their cellular constituents. This process is activated by various stressors, including nutrient deprivation and DNA damage, and has also been associated with tumor progression. In the present study, we aimed to determine the expression patterns, clinicopathological significance, and prognostic value of the autophagy marker microtubule-associated protein 1 light chain 3 alpha (LC3A)-an essential component of autophagic vacuoles-in rectal cancer. Methods: LC3A reactivity was measured by immunohistochemistry in tumor samples from 243 patients who underwent surgery for rectal cancer. Results: Three distinct patterns of LC3A expression were identified: diffuse cytoplasmic, perinuclear, and stone-like structures (SLS). In Kaplan-Meier survival analyses, patients positive for the SLS pattern of LC3A staining in the tumor periphery (TP) had worse overall survival (OS; p = 0.001) and disease-free survival (DFS; p = 0.030) than those without SLSs in this region, as determined by the log-rank test. When patients were stratified by tumor stage, this result was significant in those with stage T3-T4 (OS, p < 0.001; DFS, p = 0.001) but not T1-T2 disease. Multivariate Cox regression analysis further showed an association between TP-localized LC3A SLS positivity and reduced OS for the overall cohort (hazard ratio [HR] = 2.6313, 95% confidence interval [CI]: 1.090-6.349, p = 0.031) and specifically those in the T3-T4 subgroup (HR = 3.347, 95% CI: 1.657-6.760, p = 0.001). Conclusions: Our findings suggest that positivity for SLSs in the TP may hold clinical value as a biomarker for survival prognosis in rectal cancer patients.

Although cisplatin is widely used as a first-line chemotherapy agent, it has significant side effects. Herein, we synthesized a Pt(II) complex (Pt1) derived from o-vanillin-4-phenylthiosemicarbazone ligand and confirmed its crystal structure by x-ray crystallography. Complex Pt1 exhibited potent anticancer activity against various tested cancer cell lines, with particular efficacy against HepG-2 cells. Further investigations revealed that Pt1 inhibited the growth of HepG-2 cells through multiple mechanisms, including the generation of excessive reactive oxygen species (ROS), induction of DNA damage, enhancement of mitochondrial membrane permeability, promotion of apoptosis, and activation of autophagic cell death.

Prostaglandin G/H synthase 1 (PTGS1) is known to regulate platelet function and inflammation. However, its role in atrial fibrillation (AF)-related thrombosis is not well understood. This study investigates the role of PTGS1 in AF-associated thrombus formation and its underlying mechanisms.

Left atrial appendage (LAA) tissues were collected from 48 patients undergoing valve replacement surgery, divided into three groups: sinus rhythm (SR), AF with thrombus [AF (+) T (+)], and AF without thrombus [AF (+) T (-)]. PTGS1 expression, platelet activation markers (MPA, sCD40L, and d-dimer), macrophage phenotypes (M1 and M2), inflammatory cytokines (IL-1β, TNF-α, IL-6), and autophagy-related proteins (LC3II and p62) were assessed. Furthermore, the effect of PTGS1 manipulation on autophagy in endocardial endothelial cells (EECs) was examined using cell transfection experiments.

PTGS1 expression was significantly higher in LAA tissues of AF (+) T (+) patients compared to AF (+) T (-) and SR groups. It was positively correlated with reduced LAA emptying velocity (LAAEV), higher CHA2DS2-VASc scores, and elevated platelet activation markers (MPA, sCD40L, and d-dimer). Data also showed increased M1 macrophage infiltration and higher pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) in AF (+) T (+) patients, with PTGS1 expression strongly linked to these markers. Furthermore, PTGS1 overexpression inhibited autophagy in EECs by decreasing LC3II/LC3I ratio and increasing p62 levels, while PTGS1 knockdown promoted autophagy, protecting against endothelial dysfunction.

PTGS1 is overexpressed in AF patients with thrombosis and may play an important role in promoting thrombus formation through enhanced platelet activation, inflammation, and inhibition of autophagy.

Alzheimer's disease (AD) is less prevalent in men than in women, although mechanisms remain unclear. Microglia degrade aggregated amyloid β (Aβ) through the lysosomal system, including autophagy. G protein-coupled receptor family C group 6 member A (GPRC6A), predominantly expressed in mouse microglial MG6 cells, is a primary mediator of testosterone signaling. This study examines testosterone's role in modulating Aβ-induced autophagy in microglia. Testosterone promotes Aβ-induced autophagy leading to Aβ clearance in MG6 cells by suppressing extracellular signal-regulated kinase (ERK) phosphorylation and subsequently inhibiting mammalian target of rapamycin (mTOR) activation, which is abrogated by shRNA knockdown of GPRC6A. In in vivo experiments with male 5xFAD AD model mice, Aβ clearance activity is associated with autophagy in microglia and is reduced by orchiectomy, but restored by testosterone supplementation. ERK phosphorylation in the brains of male AD model mice is upregulated by orchiectomy. Therefore, testosterone is involved in autophagy-mediated Aβ clearance in microglia. Aβ accumulation in human brain samples from patients with AD is significantly lower in men than in women, with less pronounced colocalization of Aβ with p62 aggregates, suggesting enhanced autophagic activity in men. In conclusion, testosterone enhances Aβ-induced autophagy in microglia, possibly contributing to lower susceptibility to AD in men.

The phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) is the primary cause of citrus Huanglongbing (HLB) and is transmitted by the Asian citrus psyllid, Diaphorina citri. MicroRNAs (miRNAs) are key posttranscriptional regulators involved in various biological processes, yet their role in D. citri's response to CLas infection remains unclear. In this study, we found that autophagy levels were significantly elevated in CLas-infected D. citri compared to non-infected individuals. Modulating autophagy influenced CLas titers, suggesting its role in pathogen suppression. Small RNA sequencing identified differentially expressed miRNAs, with miR-278-3p being significantly downregulated by 72.3%. Functional analyses revealed that miR-278-3p regulates autophagy by targeting ATG16L1. Inhibition of miR-278-3p increased autophagosome formation, whereas its overexpression suppressed autophagy. Dual-luciferase reporter assays confirmed miR-278-3p directly binds to the 3'UTR of ATG16L1, negatively regulating its expression. Notably, miR-278-3p inhibition reduced CLas titers by 48.7%, demonstrating its role in pathogen defense. These findings provide novel insights into the molecular mechanisms underlying insect-pathogen interactions and highlight the potential of miRNA-based strategies for controlling plant diseases transmitted by insect vectors. Understanding these regulatory pathways may lead to innovative pest and disease management approaches in agriculture.

Huntington disease's (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine region (PolyQ) within the huntingtin protein (HTT). Mutated huntingtin (mHTT) is cytotoxic, particularly for striatal medium spiny neurons (MSNs), whose degeneration is the hallmark of HD. Autophagy inducers currently available promote the clearance of toxic proteins. However, due to their low selectivity and the possibility that prolonged autophagy hampers essential processes in unaffected cells, researchers have questioned their benefits in neurodegenerative diseases. Since MSNs express dopamine receptors D2 (DRD2) and D3 (DRD3) and DRD2/DRD3 agonists may activate autophagy, here, we explored how healthy and mHTT-challenged cells respond to prolonged DRD2/DRD3 agonist treatment. Autophagy activation and its effects on mHTT/polyQ clearance were studied in R6/1 mice (a genetic model of HD), their wild-type littermates, and DRD2- and DRD3-HEK cells expressing a pathogenic (Q74) and a non-pathogenic (Q23) polyQ fragment of mHTT treated with the DRD2/DRD3 agonist pramipexole. Two forms of DRD3-mediated autophagy were found: a transient mTORC1-dependent in WT mice and Q23-DRD3-HEK cells and a persistent AMPK-ULK1-activated in R6/1 mice and Q74-DRD3-HEK cells. This also promoted a robust clearance of soluble mHTT/polyQ and neuroprotection in striatal neurons and DRD3-HEK cells. The findings indicate that DRD3-induced autophagy may be a safe, disease-modifying intervention in HD patients.

Autophagy is a lysosome-mediated catabolic pathway that is dependent on the mammalian target of rapamycin (mTOR). It plays a crucial role in the degradation of aged organelles and macromolecules. Several studies have explored the role of autophagy in embryonic genome activation and its significance during the early preimplantation development of mammals. In our study, we showed that autophagy is inhibited in one-cell stage SCNT embryos when compared to fertilized counterparts in goats. Notably, we found that 6-DMAP, a kinase inhibitor, reduces the phosphorylation of ERK1/2.This reduction correlates with a decrease in autophagy levels, as indicated by the presence of LC3 puncta in 6-DMAP treated embryos. To address the inhibition of autophagy in goat SCNT embryos, we induced autophagy using Rapamycin at concentrations of 10 and 100 nM for 6 hours, immediately following chemical activation. This induction led to a significant improvement in the development of goat SCNT embryos, as evidenced by an increased blastocyst rate compared to the control group. Our findings suggest that the induction of autophagy during early hours of one-cell stage embryos is critical for pre-implantation development in goat SCNT embryos warrant further investigation. This research opens new avenues for understanding the role of autophagy in embryonic development and its applications in reproductive biotechnology.

The aim of this study was to investigate the effects of the antidepressant tianeptine on the mechanistic target of rapamycin complex 1(mTORC1)-mediated autophagy pathway in primary hippocampal neurons exposed to B27-deprived conditions. When primary hippocampal neurons were treated with tianeptine at doses of 1, 10, 50, and 100 µM for 3 days under B27-deprived conditions, we observed that it activated autophagy and increased the formation of autophagosomes through the upregulation of autophagic proteins, including autophagy-activating kinase 1 (ULK1), Beclin 1, LC3B-II/I, and p62. And at a concentration of 100 µM tianeptine, the decrease in mTORC1 phosphorylation induced by B27 deprivation was significantly reversed. Changes in the expression of autophagic proteins induced by B27 deprivation were reversed by tianeptine treatment in a concentration-dependent manner, and tianeptine significantly reduced the increase in LC3B membrane number induced by B27 deprivation, an effect that was blocked by pretreatment with rapamycin. In conclusion, tianeptine attenuated the activity of mTORC1-mediated autophagy in primary rat hippocampal neurons under B27-deprived conditions. These results may suggest a novel mechanism by which tianeptine may affect autophagy in neurons.

MicroRNAs (miRNAs) are double-edged swords in hepatocellular carcinoma (HCC) that play a dual role in disease progression and suppression. The pivotal role of miRNAs in gene regulation emphasizes their potential to disrupt critical cellular processes, including mitochondrial function. Given the indispensable role of mitochondria in energy production, apoptosis, and metabolic control, all of which are central to HCC progression, understanding the miRNA-mitochondria axis is crucial. MiRNAs emerge as pivotal regulators of mitochondrial function, exerting profound influence over HCC progression. This comprehensive review delves into the multifaceted roles of miRNAs in modulating mitochondrial biogenesis, dynamics, and apoptosis. MiRNA impacts key metabolic pathways, including energy metabolism, fatty acid metabolism, and oxidative stress. The intricate interplay between miRNAs and mitochondrial function extends to the regulation of mitophagy and ferroptosis. By exploring the microRNA-mitochondrial axis, this review offers insights for identifying novel diagnostic and therapeutic targets.

The intestine is an important immune organ of aquatic animals and it plays an essential role in maintaining body health and anti-oxidative stress. To investigate the toxic effects of deltamethrin in intestinal tissue of Chinese mitten crabs (Eriocheir sinensis), 120 healthy crabs were randomly divided into two experimental groups (blank control group and deltamethrin-treated group), with three replicates in each group. After being treated with deltamethrin for 24 h, 48 h, 72 h, and 96 h, intestinal tissues were collected aseptically to assess the effects of deltamethrin on oxidative stress, immunity, apoptosis-related genes, and the structure of microflora in intestinal tissues. Additionally, correlations between gut microbiota composition and intestinal tissue damage-associated genes were analyzed. The results demonstrated that prolonged exposure to deltamethrin induced oxidative stress damage in intestinal tissue. Compared with the blank control group, the expression of autophagy-related genes B-cell lymphoma/Leukemia-2 (bcl-2), c-Jun N-terminal kinase (jnk), Microtuble-associated protein light chain 3 (lc3c), Cysteine-dependent Aspartate-specific Protease 8 (caspase 8), BECN1(beclin1), oxidative stress damage-related genes MAS1 proto-oncogene (mas), Glutathione Peroxidase (gpx), kelch-like ECH-associated protein 1 (keap1), Sequestosome 1 (p62), Interleukin-6 (il-6), and immune-related genes Lipopolysaccharide-induced TNF-alpha Factor (litaf), Heat shock protein 90 (hsp90) and prophenoloxidase (propo) in the deltamethrin treatment group were significantly up-regulated at 96 h (p < 0.05 or p < 0.01). Additionally, 16S rRNA sequencing showed that the diversity of intestinal flora in the deltamethrin-treated group was significantly higher compared with the blank control group (p < 0.01). Analysis of the differences in the composition of intestinal flora at the genus level showed that the relative abundance of Candidatus Bacilloplasma in the deltamethrin treatment group was significantly lower than that in the blank control group (p < 0.01). In contrast, the relative abundances of Flavobacterium, Lachnospiraceae_NK4A136_group, Acinetobacter, Chryseobacterium, Lacihabitans, Taibaiella, Hydrogenophaga, Acidovorax, and Undibacterium were significantly higher than those in the blank control group (p < 0.05 or p < 0.01). Pearson correlation analysis revealed that Malaciobacter, Shewanella, and Prevotella exhibited significant positive correlations with gene indicators (jnk, gpx, lc3c, litaf, hsp90), while Dysgonomonas, Vibrio, and Flavobacterium demonstrated significant negative correlations with multiple gene indicators (caspase 8, p62, il-16, keap1, jnk, etc). These results demonstrate that deltamethrin significantly impacts the gut microbiota, immune function, and antioxidant capacity of E. sinensis. The changes in gut microbiota have correlations with the biomarkers of intestinal tissue injury genes, indicating that gut microbiota plays a crucial role in deltamethrin-induced intestinal tissue damage. These insights contribute to a better understanding of the ecological risks associated with deltamethrin exposure in aquatic organisms.

After ingestion into macrophage phagosomes, some bacterial pathogens such as Mycobacterium tuberculosis ( Mtb ) evade killing by preventing phagosome acidification and fusion of the phagosome with a lysosome. Mtb accumulates extracellular polyphosphate (polyP), and polyP inhibits macrophage phagosome acidification and bacterial killing. In Dictyostelium discoideum , polyP also inhibits bacterial killing, and we identified some proteins in D. discoideum that polyP requires to suppress the killing of ingested bacteria. Here, we find that pharmacological inhibition of human orthologues of the D. discoideum proteins, including P2Y1 receptors, mammalian Target of Rapamycin (mTOR), and inositol hexakisphosphate kinase, enhances the killing of Mtb , Legionella pneumophila , and Listeria monocytogenes by human macrophages. Mtb inhibits phagosome acidification, expression of the proinflammatory marker CD54, and autophagy, and increases expression of the anti-inflammatory marker CD206. In Mtb -infected macrophages, the polyP-degrading enzyme polyphosphatase (ScPPX) and inhibitors reversed these effects, with ScPPX increasing CD54 expression more in female macrophages compared to male macrophages. In addition, Mtb inhibits proteasome activity, and some, but not all, inhibitors reversed these effects. While the existence of a dedicated polyP signaling pathway remains uncertain, our findings suggest that pharmacological inhibition of select host proteins can restore macrophage function and enhances the killing of intracellular pathogens.

Human macrophages engulf bacteria into phagosomes, which then fuse with lysosomes to kill the bacteria. However, after engulfment, pathogenic bacteria such as Mycobacterium tuberculosis , Legionella pneumophila , and Listeria monocytogenes can block phagosome-lysosome fusion, allowing their survival. Here, we show that pharmacological inhibition of specific macrophage proteins reverses these effects and enhances bacterial killing. These findings suggest that targeting host factors involved in these processes may provide a therapeutic strategy to improve macrophage function against infections such as tuberculosis, Legionnaires' disease, and listeriosis.

Hexavalent chromium [Cr(VI)], due to its high solubility and permeability, is significantly more toxic than trivalent chromium [Cr(III)] as it generates reactive oxygen species (ROS) during cellular reduction. Industrial discharges have led to increasing Cr(VI) contamination in surface and groundwater, posing serious environmental and public health concerns. In our previous study, we demonstrated that exposure to 5 ppm Cr(VI) for 4 and 8 months adversely affected body weight, water consumption, and liver function in Swiss albino mice. Histological analyses revealed tissue alterations, disrupted DNA repair gene expression in liver tissue, and a marked increase in apoptotic gene expression after 8 months of exposure. Building on these findings, we employed the same Cr(VI) concentration (5 ppm via drinking water) over 4 and 8 months in the present study. Our results showed a significant increase in ROS generation in the liver, brain, and kidney tissues at both time intervals. Additionally, the presence of autophagolysosomes was markedly elevated after chronic Cr(VI) exposure in each tissue. We also observed altered expression patterns of key autophagy-related genes (Atg5, Beclin1, and Lc3) and mTor in these tissues. Immunohistochemical analysis further confirmed a significant increase in LC3B expression after 4 months of exposure. Our findings suggest that heightened intracellular oxidative stress triggers a protective autophagy response, mediated via mTOR signaling, to maintain cellular integrity. However, prolonged toxic insult and ROS accumulation may eventually shift pro-survival autophagy toward apoptotic cell death in the liver and brain tissues.

Acrylamide (ACR) is a toxic agent for humans and animals. Gentisic acid, an aspirin metabolite, has antioxidant activity. Therefore, the present study investigated the probable protective effects of aspirin and gentisic acid on ACR-induced neurotoxicity in PC12 cells and rats. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to assess the effects of aspirin and gentisic acid (1.25, 2.5, 5 µM) on ACR (5 mM) toxicity. Male Wistar rats were randomly divided into 13 groups: (1) Control group, (2) ACR (50 mg/kg, 11 days, i.p.), (3-5) ACR + aspirin (25, 50, 75 mg/kg, 11 days, p.o.), (6-8) ACR + gentisic acid (25, 50, 75 mg/kg, 11 days, p.o.), (9) ACR + vitamin E (200 mg/kg, every other day, i.p.), (10, 11) Aspirin (75, 100 mg/kg, 11 days, p.o.), (12, 13) Gentisic acid (75, 100 mg/kg, 11 days, p.o.). Behavioral tests were assessed on the final day of the study. In the cerebral cortex, malondialdehyde (MDA), glutathione (GSH), cleaved-caspase-3, and microtubule-associated protein 1A/1B-light chain 3 (LC3) protein levels were evaluated. When compared with the ACR group, aspirin (2.5, 5 µM) and gentisic acid (2.5 µM) significantly enhanced cell viability. In comparison to the control group, ACR induced severe motor impairment, elevated MDA, cleaved-caspase-3, LC3 II/I ratio, and decreased GSH levels in the cerebral cortex of rats. ACR-induced changes were significantly reversed by aspirin and gentisic acid (25 mg/kg). Oxidative stress, apoptosis, and autophagy play important roles in the neurotoxicity of ACR. Aspirin and gentisic acid significantly reduced ACR-induced toxicity by inhibiting the mentioned mechanisms.

Prohibitin 1 (PHB1) and prohibitin 2 (PHB2) are highly conserved proteins belonging to the stomatin-prohibitin flotillin-HflC/K (SPFH) protein superfamily. They are ubiquitously expressed and implicated in the regulation of cell proliferation, migration, and survival. However, the expression and biological functions of PHB1/PHB2 in atherosclerosis (AS) remain unclear. In the present study, an enzyme-linked immunosorbent assay was used to detect PHB1/PHB2 expression in the serum of patients with hyperlipidemia. The potential effect and mechanism of PHB1/PHB2 in apolipoprotein E-deficient (ApoE-/-) mice were also investigated. shRNA-PHB1 and shRNA-PHB2 lentiviruses were engineered and tail vein-injected into ApoE-/- mice fed a high-fat diet. IL-8, a proatherogenic cytokine, was used as an inducer in vitro. The effects of a PHB1/PHB2 knockdown on vascular smooth muscle cell (VSMC) proliferation, migration, and autophagy and endothelial cell (EC) adhesion were evaluated using methyl thiazolyl tetrazolium (MTT), Transwell migration, Boyden chamber, and monocyte adhesion assays, as well as transmission electron microscopy. Compared with the healthy subjects, PHB1/PHB2 expression was elevated in the serum of patients with hyperlipidemia. Animal experiments showed that downregulation of PHBs reduced the area of atherosclerotic lesions, and the expression of cyclinD1, MMP9, and LC3. In addition, in vitro experiments showed that downregulating PHB1/PHB2 expression under inflammatory stimulation reduced the adhesion, proliferation, migration, and autophagy of ECs and VSMCs by inhibiting the PI3K/Akt/mTOR pathway activation. Collectively, our findings showed that PHBs are activly associated with AS progression.

Premature graying of hair (PGH) is a common disorder with a multifactorial etiology. Autophagy, which is self-cellular digestion, has been linked to melanin pigment formation; however, the role of autophagy in PGH has not been investigated well.

The study aimed to evaluate the relationship between PGH and autophagy by measuring gene expression and serum microtubule-associated protein light chain 3 (LC3) concentration.

A case-control study was conducted on 39 PGH patients and 21 controls. Patients clinically diagnosed with PGH and aged <30 years were included in the study. Blood samples were taken to detect LC3B protein by ELISA in the serum of both groups. White hairs from both groups were collected to detect LC3B gene expression by PCR.

There was a statistically significant difference between the two groups as regards expression levels of the LC3 gene by PCR (P<0.001), with the mean in the control group (0.71± 0.3) lower than in the PGH group (5.1 ± 1.4). Also, there was a positive significant correlation between LC3 concentration and LC3 gene expression in control (r=0.867, P< 0.001) and in PGH patients (r=0.954, P≤0.001). Multivariate logistic regression analysis for PGH predictors using age, sex (female), hemoglobin level, LC3 concentration, and LC3 gene expression revealed that the only predictor of PGH was LC3 gene expression.

Premature graying of hair may have a link with autophagy. LC3 gene expression was increased in PGH patients as compared to the control. LC3 gene expression may be an independent predictor of PGH development. Autophagy modulation may be a therapeutic target for PGH.

We studied the role of caffeine intake on endothelial function in SLE by assessing its effect on circulating endothelial progenitor cells (EPCs) both ex vivo in SLE patients and in vitro in healthy donors (HD) treated with SLE sera.

We enrolled SLE patients without traditional cardiovascular risks factors. Caffeine intake was evaluated with a 7-day food frequency questionnaire. EPCs percentage was assessed by flow cytometry analysis and, subsequently, EPCs pooled from six HD were co-cultured with caffeine with and without SLE sera. After 7 days, we evaluated cells' morphology and ability to form colonies, the percentage of apoptotic cells by flow cytometry analysis and the levels of autophagy and apoptotic markers by western blot. Finally, we performed a western blot analysis to assess the A2AR/SIRT3/AMPK pathway.

We enrolled 31 SLE patients, and observed a positive correlation between caffeine intake and circulating EPCs percentage. HD EPCs treated with SLE sera and caffeine showed an improvement in morphology and in number of EPCs colony-forming units in comparison with those incubated without caffeine. Caffeine was able to restore autophagy and apoptotic markers in HD EPCs as before SLE sera treatment. Finally, caffeine treatment was able to significantly reduce A2AR levels, leading to an increase in protein levels of SIRT3 and subsequently AMPK phosphorylation.

Caffeine intake positively correlated with the percentage of circulating EPCs in SLE patients; moreover, caffeine in vitro treatment was able to improve EPC survival and vitality through the inhibition of apoptosis and the promotion of autophagy via A2AR/SIRT3/AMPK pathway.

MAVS (mitochondrial antiviral signaling protein) is a crucial adaptor in antiviral innate immunity that must be tightly regulated to maintain immune homeostasis. In this study, we identified the duck Anas platyrhynchos domesticus TRIM13 (ApdTRIM13) as a novel negative regulator of duck MAVS (ApdMAVS) that mediates the antiviral innate immune response. Upon infection with RNA viruses, ApdTRIM13 expression increased, and it specifically binds to ApdMAVS through its TM domain, facilitating the degradation of ApdMAVS in a manner independent of E3 ligase activity. Furthermore, ApdTRIM13 recruits the autophagic cargo receptor duck SQSTM1 (ApdSQSTM1), which facilitates its interaction with ApdMAVS independent of ubiquitin signaling, and subsequently delivers ApdMAVS to phagophores for degradation. Depletion of ApdSQSTM1 reduces ApdTRIM13-mediated autophagic degradation of ApdMAVS, thereby enhancing the antiviral immune response. Collectively, our findings reveal a novel mechanism by which ApdTRIM13 regulates type I interferon production by targeting ApdMAVS for selective autophagic degradation mediated by ApdSQSTM1, providing insights into the crosstalk between selective autophagy and innate immune responses in avian species.Abbreviation: 3-MA: 3-methyladenine; ATG5: autophagy related 5; baf A1: bafilomycin A1; BECN1: beclin 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CARD: caspase recruitment domain; co-IP: co-immunoprecipitation; DEFs: duck embryonic fibroblasts; DTMUV: duck Tembusu virus; eGFP: enhanced green fluorescent protein; hpi: hours post infection; IFIH1/MDA5: interferon induced with helicase C domain 1; IFN: interferon; IKBKE/IKKε: inhibitor of nuclear factor kappa B kinase subunit epsilon; IP: immunoprecipitation; IRF7: interferon regulatory factor 7; ISRE: interferon-stimulated response element; mAb: monoclonal antibody; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MOI: multiplicity of infection; NBR1: NBR1 autophagy cargo receptor; NFKB: nuclear factor kappa B; pAb: polyclonal antibody; poly(I:C): Polyriboinosinic polyribocytidylic acid; RIGI: RNA sensor RIG-I; RLR: RIGI-like-receptor; SeV: sendai virus; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TCID50: 50% tissue culture infectious dose; TM: tansmembrane; TOLLIP: toll interacting protein; TRIM: tripartite motif containing; UBA: ubiquitin-associated domain; Ub: ubiquitin; VSV: vesicular stomatitis virus; WT: wild type.

Our multi-omics study investigated the molecular mechanisms underlying autism spectrum disorder (ASD) using Shank3Δ4-22 and Cntnap2-/- mouse models. Through global- and phospho- proteomics of the mouse cortex, we focused on shared molecular changes and found that autophagy was particularly affected in both models. Global proteomics identified a small number of differentially expressed proteins that significantly impact postsynaptic components and synaptic function, including key pathways such as mTOR signaling. Phosphoproteomics revealed unique phosphorylation sites in autophagy-related proteins such as ULK2, RB1CC1, ATG16L1, and ATG9, suggesting that altered phosphorylation patterns contribute to impaired autophagic flux in ASD. SH-SY5Y cells with SHANK3 gene deletion showed elevated LC3-II and p62 levels, indicating autophagosome accumulation and autophagy initiation, while the reduced level of the lysosomal activity marker LAMP1 suggested impaired autophagosome-lysosome fusion. The study highlights the involvement of reactive nitrogen species and nitric oxide (NO) on autophagy disruption. Importantly, inhibition of neuronal NO synthase (nNOS) by 7-NI normalized autophagy markers levels in the SH-SY5Y cells and primary cultured neurons. We have previously shown that nNOS inhibition improved synaptic and behavioral phenotypes in Shank3Δ4-22 and Cntnap2-/- mouse models. Our multi-omics study reveals differential expression and phosphorylation of autophagy-related proteins in ASD but further investigation is needed to prove the full involvement of autophagy in ASD. Our study underscores the need for further examination into the functional consequences of the identified phosphorylation sites, which may offer potential novel therapeutic autophagy-related targets for ASD treatment.