Key Autophagy Genes Research Articles

An update on autophagy disorders.

Macroautophagy is a highly conserved cellular pathway for the degradation and recycling of defective cargo including proteins, organelles, and macromolecular complexes. As autophagy is particularly relevant for cellular homeostasis in post-mitotic tissues, congenital disorders of autophagy, due to monogenic defects in key autophagy genes, share a common "clinical signature" including neurodevelopmental, neurodegenerative, and neuromuscular features, as well as variable abnormalities of the eyes, skin, heart, bones, immune cells, and other organ systems, depending on the expression pattern and the specific function of the defective proteins. Since the clinical and genetic resolution of EPG5-related Vici syndrome, the paradigmatic congenital disorder of autophagy, the widespread use of massively parallel sequencing has resulted in the identification of a growing number of autophagy-associated disease genes, encoding members of the core autophagy machinery as well as related proteins. Recently identified monogenic disorders linking selective autophagy, vesicular trafficking, and other pathways have further expanded the molecular and phenotypical spectrum of congenital disorders of autophagy as a clinical disease spectrum. Moreover, significant advances in basic research have enhanced the understanding of the underlying pathophysiology as a basis for therapy development. Here, we review (i) autophagy in the context of other intracellular trafficking pathways; (ii) the main congenital disorders of autophagy and their typical clinico-pathological signatures; and (iii) the recommended primary health surveillance in monogenic disorders of autophagy based on available evidence. We further discuss recently identified molecular mechanisms that inform the current understanding of autophagy in health and disease, as well as perspectives on future therapeutic approaches.

RNA-Seq analysis reveals the different mechanisms triggered by bovine and equine after infection with FMDV.

Foot-and-mouth disease virus (FMDV) is an important pathogen of the MicroRNA virus family. Infection of livestock can cause physical weakness, weight loss, reduced milk production, and a significant reduction in productivity for an extended period. It also causes a high mortality rate in young animals, seriously affecting livestock production. The host range of FMDV is mainly limited to cloven-hoofed animals such as cattle and sheep, while odd-toed ungulates such as horses and donkeys have natural resistance to FMDV. The mechanism underlying this resistance in odd-toed ungulates remains unclear. This study aimed to analyze the differences between FMDV-infected cattle and horses to provide valuable insights into the host-FMDV interaction mechanisms, thereby contributing to the control of foot-and-mouth disease and promoting the development of the livestock industry. We observed the distribution of integrins, which help FMDV enter host cells, in the nasopharyngeal tissues of cattle and horses using immunohistochemistry. Then, we employed high-throughput RNA sequencing (RNA-Seq) to study the changes in host gene expression in the nasopharyngeal epithelial tissues of cattle and horses after FMDV infection. We performed enrichment analysis of GO and KEGG pathways after FMDV infection and validated related genes through qPCR. The immunohistochemical results showed that both cattle and horses had four integrin receptors that could assist FMDV entry into host cells. The transcriptome analysis revealed that after FMDV infection, pro-apoptotic genes such as caspase-3 (CASP3) and cytochrome C (CYCS) were upregulated in cattle, while apoptosis-inhibiting genes such as NAIP and BCL2A1 were downregulated. In contrast, the expression trend of related genes in horses was opposite to that in cattle. Additionally, autophagy-related genes such as beclin 1, ATG101, ATG4B, ATG4A, ATG13, and BCL2A1 were downregulated in cattle after FMDV infection, indicating that cattle did not clear the virus through autophagy. However, key autophagy genes including ATG1, ATG3, ATG9, ATG12, and ATG16L1 were significantly upregulated in horses after viral infection. Both water buffaloes and Mongolian horses express integrin receptors that allow FMDV entry into cells. Therefore, the resistance of Mongolian horses to FMDV may result from more changes in intracellular mechanisms, including processes such as autophagy and apoptosis. Significant differences were observed between water buffaloes and Mongolian horses in these processes, suggesting that these processes influence FMDV replication and synthesis.

Autophagy is the main driver of radioresistance of HNSCC cells in mild hypoxia.

Hypoxia poses a significant challenge to the effectiveness of radiotherapy in head and neck squamous cell carcinoma (HNSCC) patients, and it is imperative to discover novel approaches to overcome this. In this study, we investigated the underlying mechanisms contributing to x-ray radioresistance in HPV-negative HNSCC cells under mild hypoxic conditions (1% oxygen) and explored the potential for autophagy modulation as a promising therapeutic strategy. Our findings show that HNSCC cells exposed to mild hypoxic conditions exhibit increased radioresistance, which is largely mediated by the hypoxia-inducible factor (HIF) pathway. We demonstrate that siRNA knockdown of HIF-1α and HIF-1β leads to increased radiosensitivity in HNSCC cells under hypoxia. Hypoxia-induced radioresistance was not attributed to differences in DNA double strand break repair kinetics, as these remain largely unchanged under normoxic and hypoxic conditions. Rather, we identify autophagy as a critical protective mechanism in HNSCC cells following irradiation under mild hypoxia conditions. Targeting key autophagy genes, such as BECLIN1 and BNIP3/3L, using siRNA sensitizes these cells to irradiation. Whilst autophagy's role in hypoxic radioresistance remains controversial, this study highlights the importance of autophagy modulation as a potential therapeutic approach to enhance the effectiveness of radiotherapy in HNSCC.

Role of autophagy-related genes in liver cancer prognosis

Autophagy, a highly conserved process of protein and organelle degradation, has emerged as a critical regulator in various diseases, including cancer progression. In the context of liver cancer, the predictive value of autophagy-related genes remains ambiguous. Leveraging chip datasets from the TCGA and GTEx databases, we identified 23 differentially expressed autophagy-related genes in liver cancer. Notably, five key autophagy genes, PRKAA2, BIRC5, MAPT, IGF1, and SPNS1, were highlighted as potential prognostic markers, with MAPT showing significant overexpression in clinical samples. In vitro cellular assays further demonstrated that MAPT promotes liver cancer cell proliferation, migration, and invasion by inhibiting autophagy and suppressing apoptosis. Subsequent in vivo studies further corroborated the pro-tumorigenic role of MAPT by suppressing autophagy. Collectively, our model based on the five key genes provides a promising tool for predicting liver cancer prognosis, with MAPT emerging as a pivotal factor in tumor progression through autophagy modulation.

ATG7(2) Interacts With Metabolic Proteins and Regulates Central Energy Metabolism.

Macroautophagy/autophagy is an essential catabolic process that targets a wide variety of cellular components including proteins, organelles, and pathogens. ATG7, a protein involved in the autophagy process, plays a crucial role in maintaining cellular homeostasis and can contribute to the development of diseases such as cancer. ATG7 initiates autophagy by facilitating the lipidation of the ATG8 proteins in the growing autophagosome membrane. The noncanonical isoform ATG7(2) is unable to perform ATG8 lipidation; however, its cellular regulation and function are unknown. Here, we uncovered a distinct regulation and function of ATG7(2) in contrast with ATG7(1), the canonical isoform. First, affinity-purification mass spectrometry analysis revealed that ATG7(2) establishes direct protein-protein interactions (PPIs) with metabolic proteins, whereas ATG7(1) primarily interacts with autophagy machinery proteins. Furthermore, we identified that ATG7(2) mediates a decrease in metabolic activity, highlighting a novel splice-dependent function of this important autophagy protein. Then, we found a divergent expression pattern of ATG7(1) and ATG7(2) across human tissues. Conclusively, our work uncovers the divergent patterns of expression, protein interactions, and function of ATG7(2) in contrast to ATG7(1). These findings suggest a molecular switch between main catabolic processes through isoform-dependent expression of a key autophagy gene.

Association of ATG10 rs1864183, ATG16L1 rs2241880 and miR-126 with esophageal cancer.

In India, esophageal cancer (EC) is among the major cause of cancer-related deaths in both sexes. In recent past, autophagy has emerged as one of the crucial process associated with cancer. In the development of EC, the role of autophagy and the precise molecular mechanism involved has yet to be fully understood. Recently, a small number of studies have proposed how variations in autophagy genes affect the growth and development of EC. Micro-RNA's are also known to play a critical role in the development of EC. Here, we examined the relationship between the risk of EC and two single-nucleotide polymorphisms (SNPs) in the key autophagy genes, ATG10 rs1864183 and ATG16L1 rs2241880. We also analyzed the association of miR-107 and miR-126 with EC as these miRNA's are associated with autophagy. A total of 230 EC patients and 230 healthy controls from North-west Indian population were enrolled. ATG10 rs1864183 and ATG16L1 rs2241880 polymorphism were analyzed using TaqMan genotyping assay. Expression levels of miR-107 and miR-126 were analyzed through quantitative PCR using SYBR green chemistry. We found significant association of CT + CC genotype (OR 0.64, p = 0.022) in recessive model for ATG10 rs1864183 polymorphism with decreased EC risk. For ATG16L1 rs2241880 polymorphism significant association for AG genotype (OR 1.48, p = 0.05) and G allele (OR 1.43, p = 0.025) was observed for increased EC risk. Expression levels of miR-126 were also found to be significantly up regulated (p = 0.008). Our results suggest that ATG10 rs1864183, ATG16L1 rs2241880 and miR-126 may be associated with esophageal carcinogenesis and warrant further investigation.

Autophagy impairment and lifespan reduction caused by Atg1 RNAi or Atg18 RNAi expression in adult fruit flies (Drosophila melanogaster).

Autophagy, an autophagosome and lysosome-based eukaryotic cellular degradation system, has previously been implicated in lifespan regulation in different animal models. In this report, we show that expression of the RNAi transgenes targeting the transcripts of the key autophagy genes Atg1 or Atg18 in adult fly muscle or glia does not affect the overall levels of autophagosomes in those tissues and does not change the lifespan of the tested flies but the lifespan reduction phenotype has become apparent when Atg1 RNAi or Atg18 RNAi is expressed ubiquitously in adult flies or after autophagy is eradicated through the knockdown of Atg1 or Atg18 in adult fly adipocytes. Lifespan reduction was also observed when Atg1 or Atg18 was knocked down in adult fly enteroblasts and midgut stem cells. Overexpression of wild-type Atg1 in adult fly muscle or adipocytes reduces the lifespan and causes accumulation of high levels of ubiquitinated protein aggregates in muscles. Our research data have highlighted the important functions of the key autophagy genes in adult fly adipocytes, enteroblasts, and midgut stem cells and their undetermined roles in adult fly muscle and glia for lifespan regulation.

Genome-wide identification and characterization of the PbrATG family in Pyrus bretschneideri and functional analysis of PbrATG1a in response to Botryosphaeria dothidea

The pear is an economic fruit that is widely planted around the world and is loved by people for its rich nutritional value. Autophagy is a self-protection mechanism in eukaryotes, and its occurrence often accompanied by the degradation of damaged substances in cells and the recycling of nutrients. Autophagy is one of the mechanisms through which plants respond to environmental stress and plays an important role in plant development and stress resistance. Functional studies of autophagy-related genes (ATGs) have been performed on a variety of plant species, but little information is available on the ATG family in pear (Pyrus bretschneideri Rehd). Therefore, we analyzed the evolutionary dynamics and performed a genome-wide characterization of the PbrATG gene family. A total of 28 PbrATG members were identified. Phylogenetic analysis showed that PbrATGs were more closely related to ATGs of European pear and apple. Evolutionary analysis revealed that whole-genome duplication (WGD) and dispersed duplication events were the main driving forces of PbrATG family expansion. Expression analysis of different pear tissues showed that all the genes were expressed in different pear tissues, and different PbrATGs are expressed at different times and in different locations. Moreover, all PbrATGs also responded to different abiotic stresses, especially salt and drought stress, which elicited the highest expression levels. Pear seedlings were subsequently infected with Botryosphaeria dothidea (B. dothidea). The results showed that different PbrATGs had different expression patterns at different infection stages. According to the gene expression data, PbrATG1a was selected as a key autophagy gene for further analysis. Silencing of PbrATG1a reduced the resistance of pear to B. dothidea, which resulted in increased lesions, reactive oxygen species (ROS) contents, antioxidant enzyme activity, and gene expression levels in the silenced pear seedlings after B. dothidea inoculation. In this study, a comprehensive bioinformatic analysis of ATGs was conducted, and the functions of PbrATGs in pear development and in response to stress were elucidated, which laid a foundation for further study of the molecular mechanism of autophagy and a new strategy for pear resistance breeding.

Alternative splicing regulates autophagy in response to environmental stresses in cucumber (Cucumis sativus)

Autophagy is important for cell survival during stress and nutrient recycle. However, the mechanisms involved in regulating autophagy in cucumber have not been determined. Here, we analyzed the transcript abundance of key autophagy genes in the conjugation pathway including ATG3, ATG4, ATG5, ATG7, ATG8, ATG10, ATG12 and ATG16 in response to nitrogen deficiency, pathogen infection and oxidative stress in Cucumis sativus. ATG8 protein abundance was analyzed using immunoblot analysis. Seven out of 13 autophagy genes studied have splice variances including ATG3, ATG4, ATG5, ATG8b, ATG8c, ATG8e and ATG10. All except ATG5 have changes in transcript variances abundance upon nitrogen starvation, indicating regulation via alternative splicing is pervasive in autophagy genes. This is the first report of alternative splicing in autophagy genes in cucumbers. However, transcription control is also important, especially in ATG4, and those without transcript variances. Moreover, different environment cues regulate autophagy genes through different mechanisms. The ATG8 protein tag was also regulated at the protein level through post-translational modification and blockage of degradation. This work shows that the regulation of autophagy in cucumber is complex and involves many mechanisms. Better understanding of autophagy regulation would thus help breeding plants that perform better even under environmental stresses.

The use of multiple datasets to identify autophagy-related molecular mechanisms in intracerebral hemorrhage.

Background: Intracerebral hemorrhage (ICH) is a stroke syndrome with high mortality and disability rates, but autophagy's mechanism in ICH is still unclear. We identified key autophagy genes in ICH by bioinformatics methods and explored their mechanisms. Methods: We downloaded ICH patient chip data from the Gene Expression Omnibus (GEO) database. Based on the GENE database, differentially expressed genes (DEGs) for autophagy were identified. We identified key genes through protein-protein interaction (PPI) network analysis and analyzed their associated pathways in Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). Gene-motif rankings, miRWalk and ENCORI databases were used to analyze the key gene transcription factor (TF) regulatory network and ceRNA network. Finally, relevant target pathways were obtained by gene set enrichment analysis (GSEA). Results: Eleven autophagy-related DEGs in ICH were obtained, and IL-1B, STAT3, NLRP3 and NOD2 were identified as key genes with clinical predictive value by PPI and receiver operating characteristic (ROC) curve analysis. The candidate gene expression level was significantly correlated with the immune infiltration level, and most of the key genes were positively correlated with the immune cell infiltration level. The key genes are mainly related to cytokine and receptor interactions, immune responses and other pathways. The ceRNA network predicted 8,654 interaction pairs (24 miRNAs and 2,952 lncRNAs). Conclusion: We used multiple bioinformatics datasets to identify IL-1B, STAT3, NLRP3 and NOD2 as key genes that contribute to the development of ICH.

Impaired hepatic autophagy exacerbates hepatotoxin induced liver injury

Hepatotoxins activate the hepatic survival pathway, but it is unclear whether impaired survival pathways contribute to liver injury caused by hepatotoxins. We investigated the role of hepatic autophagy, a cellular survival pathway, in cholestatic liver injury driven by a hepatotoxin. Here we demonstrate that hepatotoxin contained DDC diet impaired autophagic flux, resulting in the accumulation of p62-Ub-intrahyaline bodies (IHBs) but not the Mallory Denk-Bodies (MDBs). An impaired autophagic flux was associated with a deregulated hepatic protein-chaperonin system and significant decline in Rab family proteins. Additionally, p62-Ub-IHB accumulation activated the NRF2 pathway rather than the proteostasis-related ER stress signaling pathway and suppressed the FXR nuclear receptor. Moreover, we demonstrate that heterozygous deletion of Atg7, a key autophagy gene, aggravated the IHB accumulation and cholestatic liver injury. Conclusion: Impaired autophagy exacerbates hepatotoxin-induced cholestatic liver injury. The promotion of autophagy may represent a new therapeutic approach for hepatotoxin-induced liver damage.

The mTORC1–G9a–H3K9me2 axis negatively regulates autophagy in fatty acid–induced hepatocellular lipotoxicity

Defective autophagy and lipotoxicity are the hallmarks of nonalcoholic fatty liver disease. However, the precise molecular mechanism for the defective autophagy in lipotoxic conditions is not fully known. In the current study, we elucidated that activation of the mammalian target of rapamycin complex 1 (mTORC1)-G9a-H3K9me2 axis in fatty acid-induced lipotoxicity blocks autophagy by repressing key autophagy genes. The fatty acid-treated cells show mTORC1 activation, increased histone methyltransferase G9a levels, and suppressed autophagy as indicated by increased accumulation of the key autophagic cargo SQSTM1/p62 and decreased levels of autophagy-related proteins LC3II, Beclin1, and Atg7. Our chromatin immunoprecipitation analysis showed that decrease in autophagy was associated with increased levels of the G9a-mediated repressive H3K9me2 mark and decreased RNA polymerase II occupancy at the promoter regions of Beclin1 and Atg7 in fatty acid-treated cells. Inhibition of mTORC1 in fatty acid-treated cells decreased G9a-mediated H3K9me2 occupancy and increased polymerase II occupancy at Beclin1 and Atg7 promoters. Furthermore, mTORC1 inhibition increased the expression of Beclin1 and Atg7 in fatty acid-treated cells and decreased the accumulation of SQSTM1/p62. Interestingly, the pharmacological inhibition of G9a alone in fatty acid-treated cells decreased the H3K9me2 mark at Atg7 and Beclin1 promoters and restored the expression of Atg7 and Beclin1. Taken together, our findings have identified the mTORC1-G9a-H3K9me2 axis as a negative regulator of the autophagy pathway in hepatocellular lipotoxicity and suggest that the G9a-mediated epigenetic repression is mechanistically a key step during the repression of autophagy in lipotoxic conditions.

Autophagy Facilitates Cancer-Intrinsic Evasion of CAR T Cell-Mediated Cytotoxicity in B-Cell Malignancies

Chimeric antigen receptor T (CAR T) therapy has revolutionized clinic outcomes of patients with hematologic malignancies, but therapeutic failure due to cancer resistance and relapse remains one of the major obstacles faced by CAR T cell therapy. We herein performed an unbiased CRISPR/Cas9 loss-of-function screening, which showed that enrichment of autophagy genes (ATG3, BECN1, RB1CC1 etc.) provided protection of cancer cells from CD19 CAR T cell-mediated cytotoxicity. Bulk RNA sequencing of clinical samples also suggested the clinical relevance of autophagy to therapeutic response and relapse in patients receiving CAR T cell therapy, indicating that autophagy may promote resistance to CAR-mediated cytotoxicity of B cell malignancies. The addition of autophagy inhibitor (autophinib) during the co-culture with B cell malignancy cells significantly enhanced the killing effect of CD19 CAR T cells, whereas the addition of autophagy inducer (rapamycin) demonstrated the opposite effects. As expected, consistent results were observed upon knockout of key autophagy genes, further validating the protective role of autophagy. Pharmacological inhibition of autophagy induced caspase 8 and caspase 9 cleavage when co-cultured with CD19 CAR T cells, and the knockout of BECN1 and RB1CC1 also exhibited a substantial increase in the apoptosis. The induced apoptosis after autophagy inhibition could be obviously suppressed at the presence of TNFRSF1A knockout, which indicated that the protective effect of autophagy in the context of CAR T cell killing was mediated primarily through inhibition of TNF-α-induced apoptosis. Despite the suppressed resistance to CD19 CAR T-driven killing observed in vitro cytotoxicity assays, we also observed consistent inhibition of tumor growth with the treatment of SAR405. Mice engrafted with RB1CC1-KO Raji-luc cells demonstrated attenuated resistance to CD19 CAR T cells in vivo. Remarkably, the increased infiltration of total CD3+ T and CD19 CAR T cells into Raji-luc lymphoma tumors treated with SAR405 was further confirmed by flow cytometry and immunohistochemistry staining. The cytokine profiling data identified that CXCL10 and CXCL11 were released, at least in part, from the tumor in response to autophagy inhibition. Using existing ChIP-seq datasets in ChIP-Atlas, we found that STAT1 and IRF1 had binding peaks in the promoter region of CXCL10 and CXCL11. Additionally, autophinib and SAR405 failed to induce CXCL10 and CXCL11 mRNA and protein expression in the absence of STAT1 or IRF1. Therefore, our data further validated the role of STAT1/IRF1 induced chemokine activation by autophagy targeting in reversing tumor immune escape. In conclusion, this study offered a novel mechanism in which autophagy mediates cancer-intrinsic biology contributing to observed therapeutic failure during CAR T cell therapy, wherein both STAT1/IRF1-induced chemokine signaling activation and TNF-α induced apoptosis are involved (Figure 1). Our findings promote better understanding of the role of autophagy in cancer cell resistance to CAR T cells, which will help to identify crucial areas requiring further research to improve patient outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Folium Hibisci Mutabilis extract, a potent autophagy enhancer, exhibits neuroprotective properties in multiple models of neurodegenerative diseases

BackgroundProtein aggregates are considered key pathological features in neurodegenerative diseases (NDs). The induction of autophagy can effectively promote the clearance of ND-related misfolded proteins. ObjectiveIn this study, we aimed to screen natural autophagy enhancers from traditional Chinese medicines (TCMs) presenting potent neuroprotective potential in multiple ND models. MethodsThe autophagy enhancers were broadly screened in our established herbal extract library using the transgenic Caenorhabditis elegans (C. elegans) DA2123 strain. The neuroprotective effects of the identified autophagy enhancers were evaluated in multiple C. elegans ND models by measuring Aβ-, Tau-, α-synuclein-, and polyQ40-induced pathologies. In addition, PC-12 cells and 3 × Tg-AD mice were employed to further validate the neuroprotective ability of the identified autophagy enhancers, both in vitro and in vivo. Furthermore, RNAi bacteria and autophagy inhibitors were used to evaluate whether the observed effects of the identified autophagy enhancers were mediated by the autophagy-activated pathway. ResultsThe ethanol extract of Folium Hibisci Mutabilis (FHME) was found to significantly increase GFP::LGG-1-positive puncta in the DA2123 worms. FHME treatment markedly inhibited Aβ, α-synuclein, and polyQ40, as well as prolonging the lifespan and improving the behaviors of C. elegans, while siRNA targeting four key autophagy genes partly abrogated the protective roles of FHME in C. elegans. Additionally, FHME decreased the expression of AD-related proteins and restored cell viability in PC-12 cells, which were canceled by cotreatment with 3-methyladenine (3-MA) or bafilomycin A1 (Baf). Moreover, FHME ameliorated AD-like cognitive impairment and pathology, as well as activating autophagy in 3 × Tg-AD mice. ConclusionFHME was successfully screened from our natural product library as a potent autophagy enhancer that exhibits a neuroprotective effect in multiple ND models across species through the induction of autophagy. These findings offer a new and reliable strategy for screening autophagy inducers, as well as providing evidence that FHME may serve as a possible therapeutic agent for NDs.

Integrated analysis of mRNA and microRNA transcriptome related to immunity and autophagy in shrimp hemocytes infected with Spiroplasma eriocheiris

In recent years, the industry in charge of the cultivation of Macrobrachium nipponense (M.nipponense) has suffered significant economic losses due to an infectious pathogen called Spiroplasma eriocheiris (S.eriocheiris). There has therefore been a need to identify the key immune and autophagy genes that respond to M.nipponense's infection with S. eriocheiris to analyze its immune response mechanism and the regulation of related microRNAs (miRNAs). In this study, the mRNA and miRNA transcriptome of M.nipponense's hemocytes were analyzed at different stages of infection. This analysis employed the second and third-generation sequencing technologies. In the mRNA transcriptome, 1656 genes were expressed in healthy and susceptible M.nipponense. 892 of these were significantly up-regulated, while 764 were down-regulated. 118 genes with significant differences in autophagy, endocytosis, lysosome, Toll, IMD, and VEGF pathways were obtained from the transcriptome. In the miRNA transcriptome, 312 miRNAs (Conserved: 112, PN-type: 18, PC-type: 182) were sequenced. 74 were significantly up-regulated, and 57 were down-regulated. There were 25 miRNAs involved in regulating the Toll and IMD pathways, 41 in endocytosis, 30 in lysosome, and 12 in the VEGF pathway. An integrated analysis of immune-related miRNAs and mRNAs showed that miRNAs with significant differences (P < 0.05) such as ame-miR-29b-3p, dpu-miR-1and PC-3p-945_4074, had corresponding regulatory relationships with 118 important immune genes such as Relish, Dorsal, Caspase-3, and NF-κB. This study obtained the key immune and autophagy-related genes and corresponding regulatory miRNAs in M. nipponense's hemocytes in response to an infection by S.eriocheiris. The results can provide vital data that further reveals the defense mechanism of M.nipponense's immune system against S.eriocheiris. It can also help further comprehension and interpretation of M.nipponense's resistance mechanism to the invading S.eriocheiris, and provide molecular research information for the realization of host-directed therapies (HDT) for M.nipponense.

Unravelling the In Vitro and In Vivo Anti-Helicobacter pylori Effect of Delphinidin-3-O-Glucoside Rich Extract from Pomegranate Exocarp: Enhancing Autophagy and Downregulating TNF-α and COX2.

Fruits containing antioxidants, e.g., anthocyanins, exhibit antimicrobial activities. The emergence of drug resistance represents a major challenge in eradicating H. pylori. The current study aims to explore the effect of pomegranate exocarp anthocyanin methanol extract (PEAME) against H. pylori isolates recovered from antral gastric biopsies. The UPLC-PDA-MS/MS and 1H NMR analyses indicated delphinidin-3-O-glucoside as the major anthocyanin in the extract. The PEAME showed activity against all tested resistant isolates in vitro recording minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 128 and 256 µg/mL, respectively. In vivo investigation included evaluation of the rat gastric mucosa for malondialdehyde (MDA), catalase activity, COX2, TNF-α, and key autophagy gene expression. The combination of pomegranate with metronidazole markedly reduced the viable count of H. pylori and the level of COX2, with alleviation of H. pylori-induced inflammation and oxidative stress (reduction of MDA, p-value < 0.001; and increase in catalase activity, p-value < 0.001). Autophagy gene expression was significantly upregulated upon treatment, whereas TNF-α was downregulated. In conclusion, we comprehensively assessed the effect of PEAME against H. pylori isolates, suggesting its potential in combination with metronidazole for eradication of this pathogen. The beneficial effect of PEAME may be attributed to its ability to enhance autophagy.

Robustness of the Autophagy Pathway to Somatic Copy Number Losses

Autophagy allows cells to temporarily tolerate energy stress by replenishing critical metabolites through self-digestion, thereby attenuating the cytotoxic effects of anticancer drugs that target tumor metabolism. Autophagy defects could therefore mark a metabolically vulnerable cancer state and open a therapeutic window. While mutations of autophagy genes (ATGs) are notably rare in cancer, haploinsufficiency network analyses across many cancers have shown that the autophagy pathway is frequently hit by somatic copy number losses of ATGs such as MAP1LC3B/ATG8F (LC3), BECN1/ATG6 (Beclin-1), and ATG10. Here, we used CRISPR/Cas9 technology to delete increasing numbers of copies of one or more of these ATGs in non-small cell lung cancer cells and examined the effects on sensitivity to compounds targeting aerobic glycolysis, a hallmark of cancer metabolism. Whereas the complete knockout of one ATG blocked autophagy and led to profound metabolic vulnerability, this was not the case for combinations of different nonhomozygous deletions. In cancer patients, the effect of ATG copy number loss was blunted at the protein level and did not lead to the accumulation of p62 as a sign of reduced autophagic flux. Thus, the autophagy pathway is shown to be markedly robust and resilient, even with the concomitant copy number loss of key autophagy genes.

Quercetin protects against palmitate-induced pancreatic β-cell apoptosis by restoring lysosomal function and autophagic flux

Quercetin, a natural flavonoid, has been reported to prevent pancreatic β-cell apoptosis in animal models of diabetes. However, the underlying mechanism remains unclear. We investigated the mechanisms through which quercetin protects β cells from palmitate-induced apoptosis and determined whether autophagy is involved in this process. We found that quercetin treatment partially reduced palmitate-induced β-cell apoptosis. This protective effect was abolished by pharmacologic inhibition of autophagy and by silencing a key autophagy gene. Further analysis revealed that palmitate treatment promoted the expression of LC3 II, a marker of autophagosomes, but resulted in the blockade of autophagic flux due to lysosome dysfunction. Defective lysosome accumulation can cause lysosomal membrane permeabilization and the release of cathepsins from lysosome into the cytosol that triggers apoptosis. Treatment with quercetin reversed lysosomal dysfunction and promoted autophagosome-lysosome fusion, which restored defective autophagic flux and provoked autophagy. Overall, our results indicate that lysosomal dysfunction is a major factor that contributes to β-cell apoptosis and demonstrates that quercetin improves cell survival by restoring lysosomal function and autophagic flux. This study provides new evidence regarding the anti–apoptotic mechanism of quercetin in the treatment of type 2 diabetes.

Differential effects of 5-fluorouracil and oxaliplatin on autophagy in chemoresistant colorectal cancer cells.

Macroautophagy (hereafter autophagy) is one of the adaptive pathways that contribute to cancer cell chemoresistance. Despite the fact that autophagy can both promote and inhibit cell death, there is mounting evidence that in the context of anticancer treatment, it predominantly functions as a cell survival mechanism. Therefore, silencing of key autophagy genes emerges as a potent strategy to reduce chemoresistance. Though the importance of autophagy in chemoresistance is established, the changes in autophagy in the case of acquired chemoresistance are poorly understood. In this study, we aimed to determine the changes of autophagy in the cellular model of acquired chemoresistance of colorectal cancer cell lines HCT116 and SW620, induced by 5-fluorouracil (5-FU) or oxaliplatin (OxaPt) treatment, and determine the susceptible factors for autophagy inhibition. Our results demonstrate that in the context of autophagy, 5-FU and OxaPt have different effects on HCT116 and SW620 cell lines and their chemoresistant sublines. 5-FU inhibits autophagic flux, while changes in the flux after OxaPt treatment are cell type- and dose-dependent, inducing autophagy reduction or increase. The chemoresistant subline of HCT116 cells derived by OxaPt differs from the subline derived by 5-FU treatment - it responds to OxaPt by upregulating ATG7 protein level and autophagic flux, in contrast to downregulation in cells derived by 5-FU. Moreover, 5-FU and OxaPt treatments significantly modulate protein levels of core-autophagy proteins ATG7 and ATG12. The potential effects of 5-FU and OxaPt on ATG protein levels should be taken into account to reduce chemoresistance by applying small interferingRNAs, targeting ATG proteins.

Autophagy promotes programmed cell death and corpse clearance in specific cell types of the Arabidopsis root cap

Autophagy is a conserved quality control pathway that mediates the degradation of cellular components by targeting them to the lysosomes or vacuoles.1 Autophagy has been implicated in the regulation of some regulated cell death processes in animal systems.2 However, its function in developmentally controlled programmed cell death (dPCD) in plants remains little studied and controversial.3 Some studies have reported autophagy pro-survival roles,4,5 while others have suggested pro-death functions for autophagy,6,7 calling for further detailed investigations. Here, we investigated the role of autophagy in dPCD using the Arabidopsis root cap as an accessible and genetically tractable model system.8 In Arabidopsis, dPCD is an integral part of root cap differentiation, restricting root cap organ size to the root meristem.9 The root cap consists of two distinct tissues: the proximally positioned columella that is located at the very root tip and the lateral root cap (LRC) that flanks the root meristem up to its distal end at the start of the root elongation zone.10 We show that autophagic flux strongly increased prior to dPCD execution in both root cap tissues and depends on the key autophagy genes ATG2, ATG5, and ATG7. Systemic and organ-specific mutation of these genes shows delayed PCD execution and lack of postmortem corpse clearance in the columella but no defects in dPCD execution or corpse clearance in the distal LRC. Our results reveal a high degree of cell-type specificity in autophagy functions and suggest that autophagy roles in dPCD can considerably diverge between different cell types of the same plant organ.