Immunoexpression Pattern of Autophagy-Related Proteins in Human Congenital Anomalies of the Kidney and Urinary Tract.

The purpose of this study was to evaluate the spatiotemporal immunoexpression pattern of microtubule-associated protein 1 light chain 3 beta (LC3B), glucose-regulated protein 78 (GRP78), heat shock protein 70 (HSP70), and lysosomal-associated membrane protein 2A (LAMP2A) in normal human fetal kidney development (CTRL) and kidneys affected with congenital anomalies of the kidney and urinary tract (CAKUT). Human fetal kidneys (control, horseshoe, dysplastic, duplex, and hypoplastic) from the 18th to the 38th developmental week underwent epifluorescence microscopy analysis after being stained with antibodies. Immunoreactivity was quantified in various kidney structures, and expression dynamics were examined using linear and nonlinear regression modeling. The punctate expression of LC3B was observed mainly in tubules and glomerular cells, with dysplastic kidneys displaying distinct staining patterns. In the control group's glomeruli, LAMP2A showed a sporadic, punctate signal; in contrast to other phenotypes, duplex kidneys showed significantly stronger expression in convoluted tubules. GRP78 had a weaker expression in CAKUT kidneys, especially hypoplastic ones, while normal kidneys exhibited punctate staining of convoluted tubules and glomeruli. HSP70 staining varied among phenotypes, with dysplastic and hypoplastic kidneys exhibiting stronger staining compared to controls. Expression dynamics varied among observed autophagy markers and phenotypes, indicating their potential roles in normal and dysfunctional kidney development.

Duck STING mediates antiviral autophagy directing the interferon signaling pathway to inhibit duck plague virus infection.

Migratory birds are important vectors for virus transmission, how migratory birds recognize viruses and viruses are sustained in birds is still enigmatic. As an animal model for waterfowl among migratory birds, studying and dissecting the antiviral immunity and viral evasion in duck cells may pave a path to deciphering these puzzles. Here, we studied the mechanism of antiviral autophagy mediated by duck STING in DEF cells. The results collaborated that duck STING could significantly enhance LC3B-II/I turnover, LC3B-EGFP puncta formation, and mCherry/EGFP ratio, indicating that duck STING could induce autophagy. The autophagy induced by duck STING is not affected by shRNA knockdown of ATG5 expression, deletion of the C-terminal tail of STING, or TBK1 inhibitor BX795 treatment, indicating that duck STING activated non-classical selective autophagy is independent of interaction with TBK1, TBK1 phosphorylation, and interferon (IFN) signaling. The STING R235A mutant and Sar1A/B kinase mutant abolished duck STING induced autophagy, suggesting binding with cGAMP and COPII complex mediated transport are the critical prerequisite. Duck STING interacted with LC3B through LIR motifs to induce autophagy, the LIR 4/7 motif mutants of duck STING abolished the interaction with LC3B, and neither activated autophagy nor IFN expression, indicating that duck STING associates with LC3B directed autophagy and dictated innate immunity activation. Finally, we found that duck STING mediated autophagy significantly inhibited duck plague virus (DPV) infection via ubiquitously degraded viral proteins. Our study may shed light on one scenario about the control and evasion of diseases transmitted by migratory birds.

Autophagy is involved in granulosa cell death and follicular atresia in ewe ovaries.

In mammalian ovaries, most follicles do not ovulate and are eliminated by atresia, which primarily depends on granulosa cell (GC) apoptosis. Autophagy is an alternative mechanism involved in follicle depletion in mammals through independent or tandem action with apoptosis. However, follicular autophagy has not yet been investigated in sheep; therefore, the present study aimed to investigate the involvement of autophagy in atresia among a pool of growing antral follicles in ewe ovaries. The abundance of the autophagic marker LC3B-II was determined using western blotting in GCs collected from ewe antral follicles. The antral follicles were classified as healthy or atretic based on morphological criteria and steroid measurements in follicular fluid (FF). Immunofluorescence and confocal microscopy analyses were performed on GCs to evaluate the presence of autophagic proteins and their subcellular localisation. Caspase-3 and DNA fragmentation were assessed using western blotting and TUNEL assays, respectively, in the same GC population to investigate the simultaneous apoptosis. The novel results of this study demonstrated enhanced LC3B-II protein expression in GCs of atretic follicles compared to that of healthy ones (1.3-fold increase; P = 0.0001, ANOVA), indicating a correlation between autophagy enhancement in GCs and antral follicular atresia. Autophagy, either functioning independently or in tandem with apoptosis, may be involved in the atresia of growing antral follicles in ewe ovaries because atretic GCs also showed high levels of apoptotic markers. The findings of this study might have important implication on scientific understanding of ovarian follicle dynamics.

Computational Design of Novel Cyclic Peptides Endowed with Autophagy-Inhibiting Activity on Cancer Cell Lines.

(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the nascent autophagosomes. The inhibition of LC3 maturation results in the interference of autophagy activation. (2) In this study, starting from the structure of a known LC3B binder (LIR2-RavZ peptide), we identified new LC3B ligands by applying an in silico drug design strategy. The most promising peptides were synthesized, biophysically assayed, and biologically evaluated to ascertain their potential antiproliferative activity on five humans cell lines. (3) A cyclic peptide (named Pep6), endowed with high conformational stability (due to the presence of a disulfide bridge), displayed a Kd value on LC3B in the nanomolar range. Assays accomplished on PC3, MCF-7, and A549 cancer cell lines proved that Pep6 exhibited cytotoxic effects comparable to those of the peptide LIR2-RavZ, a reference LC3B ligand. Furthermore, it was ineffective on both normal prostatic epithelium PNT2 and autophagy-defective prostate cancer DU145 cells. (4) Pep6 can be considered a new autophagy inhibitor that can be employed as a pharmacological tool or even as a template for the rational design of new small molecules endowed with autophagy inhibitory activity.

Unexpected roles for AMPK in the suppression of autophagy and the reactivation of MTORC1 signaling during prolonged amino acid deprivation.

AMPK promotes catabolic and suppresses anabolic cell metabolism to promote cell survival during energetic stress, in part by inhibiting MTORC1, an anabolic kinase requiring sufficient levels of amino acids. We found that cells lacking AMPK displayed increased apoptotic cell death during nutrient stress caused by prolonged amino acid deprivation. We presumed that impaired macroautophagy/autophagy explained this phenotype, as a prevailing view posits that AMPK initiates autophagy (often a pro-survival response) through phosphorylation of ULK1. Unexpectedly, however, autophagy remained unimpaired in cells lacking AMPK, as monitored by several autophagic readouts in several cell lines. More surprisingly, the absence of AMPK increased ULK1 signaling and MAP1LC3B/LC3B lipidation during amino acid deprivation while AMPK-mediated phosphorylation of ULK1 S555 (a site proposed to initiate autophagy) decreased upon amino acid withdrawal or pharmacological MTORC1 inhibition. In addition, activation of AMPK with compound 991, glucose deprivation, or AICAR blunted autophagy induced by amino acid withdrawal. These results demonstrate that AMPK activation and glucose deprivation suppress autophagy. As AMPK controlled autophagy in an unexpected direction, we examined how AMPK controls MTORC1 signaling. Paradoxically, we observed impaired reactivation of MTORC1 in cells lacking AMPK upon prolonged amino acid deprivation. Together these results oppose established views that AMPK promotes autophagy and inhibits MTORC1 universally. Moreover, they reveal unexpected roles for AMPK in the suppression of autophagy and the support of MTORC1 signaling in the context of prolonged amino acid deprivation. These findings prompt a reevaluation of how AMPK and its control of autophagy and MTORC1 affect health and disease.

LC3B conjugation machinery promotes autophagy-independent HIV-1 entry in CD4+ T lymphocytes.

HIV-1 entry into CD4+ T lymphocytes relies on the viral and cellular membranes' fusion, leading to viral capsid delivery in the target cell cytoplasm. Atg8/LC3B conjugation to lipids, process named Atg8ylation mainly studied in the context of macroautophagy/autophagy, occurs transiently in the early stages of HIV-1 replication in CD4+ T lymphocytes. Despite numerous studies investigating the HIV-1-autophagy interplays, the Atg8ylation impact in these early stages of infection remains unknown. Here we found that HIV-1 exposure leads to the rapid LC3B enrichment toward the target cell plasma membrane, in close proximity with the incoming viral particles. Furthermore, we demonstrated that Atg8ylation is a key event facilitating HIV-1 entry in target CD4+ T cells. Interestingly, this effect is independent of canonical autophagy as ATG13 silencing does not prevent HIV-1 entry. Together, our results provide an unconventional role of LC3B conjugation subverted by HIV-1 to achieve a critical step of its replication cycle.Abbreviations: BafA1: bafilomycin A1; BlaM: beta-lactamase; CD4+ TL: CD4+ T lymphocytes; PtdIns3K-BECN1 complex: BECN1-containing class III phosphatidylinositol 3-kinase complex; Env: HIV-1 envelope glycoproteins; HIV-1: type 1 human immunodeficiency virus; PM: plasma membrane; PtdIns3P: phosphatidylinositol-3-phosphate; VLP: virus-like particle.

Expression of LC3A, LC3B and p62/SQSTM1 autophagy proteins in hepatocellular carcinoma (HCC) tissues and the predicted microRNAs involved in the autophagy-related pathway.

BACKGROUND: Autophagy plays multifaceted roles in regulating hepatocellular carcinoma (HCC) and the mechanisms involved are under-explored. Regulatory microRNAs (miRNAs) have been reported to target autophagy proteins but their roles in HCC is not well studied. Using HCC patient tissues, this study aims to investigate the association of autophagy with several clinicopathological parameters as well as identifying the autophagy-related miRNAs and the possible pathways. METHODS AND RESULTS: Autophagy level in the HCC patient-derived cancer and non-cancer tissues was determined by immunohistochemistry (IHC) targeting SQSTM1, LC3A and LC3B proteins. Significance tests of clinicopathological variables were tested using the Fisher's exact or Chi-square tests. Gene and miRNA expression assays were carried out and analyzed using Nanostring platform and software followed by validation of other online bioinformatics tools, namely String and miRabel. Autophagy expression was significantly higher in cancerous tissues compared to adjacent non-cancer tissues. High LC3B expression was associated with advanced tumor histology grade and tumor location. Nanostring gene expression analysis revealed that SQSTM1, PARP1 and ATG9A genes were upregulated in HCC tissues compared to non-cancer tissues while SIRT1 gene was downregulated. These genes are closely related to an autophagy pathway in HCC. Further, using miRabel tool, three downregulated miRNAs (hsa-miR-16b-5p, hsa-miR-34a-5p, and hsa-miR-660-5p) and one upregulated miRNA (hsa-miR-539-5p) were found to closely interact with the abovementioned autophagy-related genes. We then mapped out the possible pathway involving the genes and miRNAs in HCC tissues. CONCLUSIONS: We conclude that autophagy events are more active in HCC tissues compared to the adjacent non-cancer tissues. We also reported the possible role of several miRNAs in regulating autophagy-related genes in the autophagy pathway in HCC. This may contribute to the development of potential therapeutic targets for improving HCC therapy. Future investigations are warranted to validate the target genes reported in this study using a larger sample size and more targeted molecular technique.

Targeting EGFR degradation by autophagosome degraders.

Several generations of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors have been developed for the treatment of non-small cell lung cancer (NSCLC) in clinic. However, emerging drug resistance mediated by new EGFR mutations or activations by pass, leads to malignant progression of NSCLC. Proteolysis targeting chimeras (PROTACs) have been utilized to overcome the drug resistance acquired by mutant EGFR, newly potent and selective degraders are still need to be developed for clinical applications. Herein, we developed autophagosome-tethering compounds (ATTECs) in which EGFR can be anchored to microtubule-associated protein-1 light chain-3B (LC3B) on the autophagosome with the assistance of the LC3 ligand GW5074. A series of EGFR-ATTECs have been designed and synthesized. Biological evaluations showed that these compounds could degrade EGFR and exhibited moderate inhibitory effects on certain NSCLC cell lines. The ATTEC 12c potently induced the degradation of EGFR with a DC50 value of 0.98 µM and a Dmax value of 81% in HCC827 cells. Mechanistic exploration revealed that the lysosomal pathway was mainly involved in this degradation. Compound 12c also exhibited promising inhibitory activity, as well as degradation efficiency in vivo. Our study highlights that EGFR-ATTECs could be developed as a new expandable EGFR degradation tool and also reveals a novel potential therapeutic strategy to prevent drug resistance acquired EGFR mutations.

Anti-influenza A (H1N1) virus effect of gallic acid through inhibition of virulent protein production and association with autophagy.

Influenza remains one of the most serious infectious diseases. Gallic acid is one of the most common and representative phenolic acids found in various plants. This is an interesting subject to explore how gallic acid could inhibit H1N1 influenza virus infection by reducing the production of virulent proteins and interrupting autophagy machinery for influenza virus replication on the host cell. Cellular viability was assessed by XTT assay. The inhibitory effects on the H1N1 influenza virus were assessed by hemagglutination assay, plaque assay, and qRT-PCR. Western blot analysis was used for detecting protein levels of M1, M2, NP, LC3B, and beclin-1. Autophagy activity was demonstrated by acridine orange staining assay. The result demonstrated that there was no cytotoxic effect of gallic acid on A549 cells, and gallic acid could restore the cellular viability of H1N1 influenza virus-infected A549 cells within the experimental concentration treatment. Moreover, gallic acid could effectively restrain viral activity of the H1N1 influenza virus. After the treatment of gallic acid, the production of virulent H1N1 influenza virus proteins, that is, M1, M2, and NP protein were reduced. As for autophagic mechanism, both of the LC3B II conversion and the level ratio of LC3B II to LC3B I were notably decreased. The acridine orange staining assay also revealed decreased accumulation of autophagosomes in H1N1 influenza virus-infected cells. In conclusion, gallic acid suppresses H1N1 influenza viral infectivity through restoration of autophagy pathway and inhibition of virulent M1, M2, and NP protein production.

Unraveling the Impact of Dab1 Gene Silencing on the Expression of Autophagy Markers in Lung Development.

The purpose of this study was to evaluate the effects of Dab1 gene silencing on the immunoexpression of light chain 3 beta (Lc3b), glucose regulating protein 78 (Grp78), heat shock cognate 71 (Hsc70), mammalian target of rapamycin (mTOR) and lysosomal-associated membrane protein 2A (Lamp2a) in the lung tissue of developing yotari (Dab1-/-) and wild-type (wt) mice. The lung epithelium and mesenchyme of the embryos at gestational days E13.5 and E15.5 were examined using immunofluorescence and semi-quantitative methods. In the pulmonary mesenchyme and epithelium, Grp78 and Lc3b of moderate fluorescence reactivity was demonstrated in wt mice for both evaluated time points, while yotari mice exhibited only epithelial reactivity for the same markers. Mild punctate expression of Hsc70 was observed for both genotypes. A significant difference was present when analyzing mTOR expression, where wt mice showed strong perinuclear staining in the epithelium. According to our data, Dab1 gene silencing may result in autophagy abnormalities, which could then cause respiratory system pathologies via defective lung cell degradation by lysosome-dependent cell elimination.

Sodium fluoride-induced autophagy of ameloblast-like cells via the p-ULk1/ATG13/LC3B pathway in vitro.

OBJECTIVE: To investigate the effects of sodium fluoride on the ameloblast and reveal the mechanism of dental fluorosis. MATERIALS AND METHODS: Mouse ameloblast-like cell line (ALC) cells were treated with various concentrations of NaF, and subjected to Incucyte, fluorescence immunoassay, transmission electron microscopy, reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot for autophagy examination, alkaline phosphatase and alizarin red staining for mineralization after osteogenic induction. RESULTS: NaF exerts a dose-dependent inhibitory effect on ALC cell growth. TEM and fluorescence immunoassay showed that 1.5 mM or higher concentrations of NaF could induce a fusion of lysosome and mitochondria, finally increasing the number of autophagosome. RT-qPCR and western blot showed that the upregulation of autophagy related gene 13 (ATG13), downregulation of phosphorylated Unc-51-like kinase 1 (p-ULK1) were found in NaF-induced autophagy of ALC cells. The knockdown of ATG13 could rescue it as well as the expression of p-ULK1 and LC3B. Besides, alizarin red staining showed that fluoride under these concentrations could promote the mineralization of ALC. CONCLUSIONS: The data show that fluoride in higher concentration can induce autophagy via the p-ULk1/ATG13/LC3B pathway of ALCs than lower ones promote mineralization in vitro, which provides insight into the function of NaF in the autophagy and mineralization of ameloblast.

Stromal interaction molecule 1/microtubule-associated protein 1A/1B-light chain 3B complex induces metastasis of hepatocellular carcinoma by promoting autophagy.

Metastasis is the leading cause of death in hepatocellular carcinoma (HCC) patients, and autophagy plays a crucial role in this process by orchestrating epithelial-mesenchymal transition (EMT). Stromal interaction molecule 1 (STIM1), a central regulator of store-operated calcium entry (SOCE) in nonexcitable cells, is involved in the development and spread of HCC. However, the impact of STIM1 on autophagy regulation during HCC metastasis remains unclear. Here, we demonstrate that STIM1 is temporally regulated during autophagy-induced EMT in HCC cells, and knocking out (KO) STIM1 significantly reduces both autophagy and EMT. Interestingly, STIM1 enhances autophagy through both SOCE-dependent and independent pathways. Mechanistically, STIM1 directly interacts with microtubule-associated protein 1A/1B-light chain 3B (LC3B) to form a complex via the sterile-α motif (SAM) domain, which promotes autophagosome formation. Furthermore, deletion of the SAM domain of STIM1 abolishes its binding with LC3B, leading to a decrease in autophagy and EMT in HCC cells. These findings unveil a novel mechanism by which the STIM1/LC3B complex mediates autophagy and EMT in HCC cells, highlighting a potential target for preventing HCC metastasis.

Hepatocyte miR-21-5p-deficiency alleviates APAP-induced liver injury by inducing PPARγ and autophagy.

Acetaminophen (APAP)-induced liver injury is one of the most frequent causes of acute liver failure worldwide. Significant increases in the levels of miRNA-21 in both liver tissues and plasma have been observed in APAP-overdosed animals and humans. However, the mechanistic effect of miRNA-21 on acute liver injury remains unknown. In this study, we generated a new hepatocyte-specific miRNA-21 knockout (miR-21-HKO) mouse line. miR-21-HKO and the background-matched sibling wild-type (WT) mice were treated with a toxic dose of APAP. Compared with WT mice, miR-21 HKO mice showed an increased survival, a reduction of necrotic hepatocytes, and an increased expression of light chain 3 beta, which suggested an autophagy activation. The expression of PPARγ was highly induced in the livers of miR-21-HKO mice after a 2-h APAP treatment, which preceded the activation of LC3B at the 12 h APAP treatment. miR-21 negatively regulated PPARγ protein expression by targeting its 3'-UTR. When PPARγ function was blocked by a potent antagonist GW9662 in miR-21-HKO mice, the autophage activation was significantly diminished, suggesting an indispensable role of PPARγ signaling pathway in miR-21-mediated hepatotoxicity. Taken together, hepatocyte-specific depletion of miRNA-21 alleviated APAP-induced hepatotoxicity by activating PPARγ and autophagy, demonstrating a crucial new regulatory role of miR-21 in APAP-mediated liver injury.

Investigating the Association between the Autophagy Markers LC3B, SQSTM1/p62, and DRAM and Autophagy-Related Genes in Glioma.

High-grade gliomas are extremely fatal tumors, marked by severe hypoxia and therapeutic resistance. Autophagy is a cellular degradative process that can be activated by hypoxia, ultimately resulting in tumor advancement and chemo-resistance. Our study aimed to examine the link between autophagy markers' expression in low-grade gliomas (LGGs) and high-grade gliomas (HGGs). In 39 glioma cases, we assessed the protein expression of autophagy markers LC3B, SQSTM1/p62, and DRAM by immunohistochemistry (IHC) and the mRNA expression of the autophagy genes PTEN, PI3K, AKT, mTOR, ULK1, ULK2, UVRAG, Beclin 1, and VPS34 using RT-qPCR. LC3B, SQSTM1/p62, and DRAM expression were positive in 64.1%, 51.3%, and 28.2% of glioma cases, respectively. The expression of LC3B and SQSTM1/p62 was notably higher in HGGs compared to LGGs. VPS34 exhibited a significant differential expression, displaying increased fold change in HGGs compared to LGGs. Additionally, it exhibited robust positive associations with Beclin1 (rs = 0.768), UVRAG (rs = 0.802), and ULK2 (rs = 0.786) in HGGs. This underscores a potential association between autophagy and the progression of gliomas. We provide preliminary data for the functional analysis of autophagy using a cell culture model and to identify potential targets for therapeutic interventions.

α-Synuclein reduces acetylserotonin O-methyltransferase mediated melatonin biosynthesis by microtubule-associated protein 1 light chain 3 beta-related degradation pathway.

Previous studies have demonstrated that α-synuclein (α-SYN) is closely associated with rapid eye movement sleep behavior disorder (RBD) related to several neurodegenerative disorders. However, the exact molecular mechanisms are still rarely investigated. In the present study, we found that in the α-SYNA53T induced RBD-like behavior mouse model, the melatonin level in the plasma and pineal gland were significantly decreased. To elucidate the underlying mechanism of α-SYN-induced melatonin reduction, we investigated the effect of α-SYN in melatonin biosynthesis. Our findings showed that α-SYN reduced the level and activity of melatonin synthesis enzyme acetylserotonin O-methyltransferase (ASMT) in the pineal gland and in the cell cultures. In addition, we found that microtubule-associated protein 1 light chain 3 beta (LC3B) as an important autophagy adapter is involved in the degradation of ASMT. Immunoprecipitation assays revealed that α-SYN increases the binding between LC3B and ASMT, leading to ASMT degradation and a consequent reduction in melatonin biosynthesis. Collectively, our results demonstrate the molecular mechanisms of α-SYN in melatonin biosynthesis, indicating that melatonin is an important molecule involved in the α-SYN-associated RBD-like behaviors, which may provide a potential therapeutic target for RBD of Parkinson's disease.

Immunohistochemical Expression of Autophagy-Related Marker (LC3B) and Stem Cell Marker (CD44) in Molecular Subtypes of Breast Cancer.

BACKGROUND: Breast cancer (BC) is among the most prevalent aggressive type of malignancy affecting females worldwide. Despite the advance in early detection and management of BC; recurrence, metastasis and mortality remains high. This may be attributed to heterogeneity of BC which explained by the presence of breast cancer stem cells (BCSCs). BCSCs is characterized by their ability of self-renewal, unlimited proliferation and their differentiation potential.  BCSCs maintain their activity through process of autophagy. Autophagy is a catabolic pathway important for maintenance of cellular hemostasis in response to different stressful conditions. Autophagy allows BCSCs to adapt to different stressful conditions. So, it protects BCSCs from cytotoxic effects of anti-cancer therapy and anticancer resistance. METHODS: Formalin-fixed paraffin embedded fifty specimens of Invasive duct carcinoma of no special type(IDC/NST) of breast was selected and immunostained with stem cell marker CD44 and autophagy related marker LC3B antibodies. Correlation with different clinicopathological, histopathological characteristics and molecular subtypes of studied specimens were evaluated. RESULTS: Both CD44 and LC3B expression were significantly associated with lymph nodal metastasis (p =0.001 and 0.010 respectively), advanced pathological stage (p= 0.045 and 0.004 respectively) and with triple negative molecular subtype of BC (p=0.044 and 0.048 respectively). Statistically positive correlation was also found between both tumor markers expression. CONCLUSION: Results of this study suggests that CD44 and LC3B expression play a role in the clinical behavior and progression of different molecular subtypes of BC.

The modulatory effect of Lactobacillus gasseri ATCC 33323 on autophagy induced by extracellular vesicles of Helicobacter pylori in gastric epithelial cells in vitro.

Helicobacter pylori has been recognized as a true pathogen, which is associated with various gastroduodenal diseases, and gastric adenocarcinoma. The crosstalk between H. pylori virulence factors and host autophagy remains challenging. H. pylori can produce extracellular vesicles (EVs) that contribute to gastric inflammation and malignancy. Some probiotic strains have been documented to modulate cell autophagy process. This study was aimed to investigate the modulatory effect of cell-free supernatant (CFS) obtained from Lactobacillus gasseri ATCC 33323 on autophagy induced by H. pylori-derived EVs. EVs were isolated from two clinical H. pylori strains (BY-1 and OC824), and characterized using transmission electron microscopy (TEM) and dynamic light scattering (DLS). The viability of AGS cells was assessed after exposure to different concentrations of H. pylori EVs, and L. gasseri CFS. Based on MTT assay and Annexin V-FITC/PI staining, 50 µg/ml of H. pylori EVs and 10 % v/v of L. gasseri CFS were used for further cell treatment experiments. Autophagy was examined using acridin orange (AO) staining, RT-qPCR analysis for autophagy mediators (LC3B, ATG5, ATG12, ATG16L1, BECN1, MTOR, and NOD1), and western blotting for LC3B expression. H. pylori EVs were detected to range in size from 50 to 200 nm. EVs of both H. pylori strains and L. gasseri CFS showed no significant effect on cell viability as compared to untreated cells. H. pylori EVs promoted the development of acidic vesicular organelles and the expression of autophagy-related genes (LC3B, ATG5, ATG12, ATG16L1, BECN1, and NOD1), and decreased the expression of MTOR in AGS cells at 12 and 24 h time periods. In addition, the production of LC3B was increased following 12 h of treatment in AGS cells. In contrast, L. gasseri CFS effectively inhibited EVs-induced autophagy, as evidenced by reduced acidic vesicular organelle formation and modulation of autophagy markers. Our study indicated that L. gasseri CFS can effectively suppress H. pylori EV-induced autophagy in AGS cells. Further investigations are required to decipher the mechanism of action L. gasseri CFS and its metabolites on autophagy inhibition induced by H. pylori.

MicroRNA Let-7a association with glycolysis-induced autophagy in locally advanced gastric cancer: Their role in prognosis and FLOT chemotherapy resistance.

Locally advanced gastric cancer (LAGC) still poses a clinical challenge despite multimodality treatment due to multidrug resistance (MDR). Recently, research suggested that autophagy and metabolic regulation may be potential anticancer targets due to their crucial roles in MDR. Let-7a participates in glycolytic and autophagic regulations which are both essential for tumor progression and resistance to therapy. This study used IHC stains; GLUT4 and LC3B to evaluate glycolysis and autophagy respectively. Moreover, mRNA Let-7a was detected by quantitative reverse transcription PCR (q-PCR) in 53 cases of LAGC. Elevated glycolysis and autophagy in LAGC tissue specimens as indicated by high GLUT4 and LC3B expression were significantly associated with adverse prognostic factors such as high pathological grade, positive nodal metastasis, and advanced T stage. Lower Let-7a levels were significantly associated with high tumor grade and advanced T stage. A significant positive correlation between GLUT4 and LC3B expression was detected. Significant inverse correlations between let7a level and IHC expression of both GLUT4 and LC3B were found. Elevated glycolysis and autophagy were significantly associated with poor overall survival (OS). Furthermore, low levels of let-7a were significantly associated with poor OS compared to high levels. Glycolysis and autophagy in LAGC were significantly associated with poor FLOT chemotherapy response. Let7a mRNA relative expression was significantly decreased in cases showing post therapy partial response and sustained disease. Multivariate analysis showed that histologic tumor type, high GLUT4 and high LC3B expression were independent factors associated with poor OS. Poor survival and post FLOT chemotherapy resistance in LAGC cases were significantly related to elevated glycolysis, elevated autophagy, and reduced Let-7a expression. Accordingly, combined therapeutic targeting of these pathways could enhance chemosensitivity in LAGC.

Autophagy indicators in oral squamous cell carcinoma.

Autophagy plays an important role in maintaining cellular homeostasis. Dysregulation of autophagy has been linked to a number of diseases, including cancer. We retrospectively evaluated immunohistochemical expression of the autophagy markers LC3B and p62 and the autophagy regulator mTOR as an indicator of autophagy in 100 surgically resected primary oral squamous cell carcinoma (OSCC) samples and sought associations with various clinicopathological factors. The expression of all three proteins was significantly higher in malignant squamous cells than in benign squamous cells in the free mucosal margin adjacent to the OSCC. Male sex, higher tumour (T) stage, node (N) stage and tumour, node, metastasis (TNM) stage were significantly associated with high marker expression; age and histological grade showed no significant association. LC3B, p62 and mTOR expression were positively correlated with one another in OSCCs, and the correlation was significant for LC3B and mTOR as well as for LC3B and p62. Disease-free survival showed an inverse correlation with high mTOR expression. Our data suggest that autophagy inhibitors and mTOR inhibitors may have a therapeutic role in the treatment of OSCCs.

Classical swine fever virus NS5A protein activates autophagy via the PP2A-DAPK3-Beclin 1 axis.

IMPORTANCE: Autophagy is a conserved degradation process that maintains cellular homeostasis and regulates native and adaptive immunity. Viruses have evolved diverse strategies to inhibit or activate autophagy for their benefit. The paper reveals that CSFV NS5A mediates the dissociation of PP2A from Beclin 1 and the association of PP2A with DAPK3 by interaction with PPP2R1A and DAPK3, PP2A dephosphorylates DAPK3 to activate its protein kinase activity, and activated DAPK3 phosphorylates Beclin 1 to trigger autophagy, indicating that NS5A activates autophagy via the PP2A-DAPK3-Beclin 1 axis. These data highlight a novel mechanism by which CSFV activates autophagy to favor its replication, thereby contributing to the development of antiviral strategies.