Despite significant improvement in the prognosis of multiple myeloma (MM), the disease remains incurable; thus, more effective therapies are required. Ribonucleoside-diphosphate reductase subunit M2 (RRM2) is significantly associated with drug resistance, rapid relapse, and poor prognosis. Previously, we found that 4-hydroxysalicylanilide (osalmid), a specific inhibitor of RRM2, exhibits anti-MM activity in vitro, in vivo, and in human patients; however, the mechanism remains unclear. Osalmid inhibits the translocation of RRM2 to the nucleus and stimulates autophagosome synthesis but inhibits subsequent autophagosome-lysosome fusion. We confirm that RRM2 binds to receptor-interacting protein kinase 3 (RIPK3) and reduces RIPK3, inhibiting autophagosome-lysosome fusion. Interestingly, the combination of osalmid and bafilomycin A1 (an autophagy inhibitor) depletes RIPK3 and aggravates p62 and autophagosome accumulation, leading to autophagic cell death. Combination therapy demonstrates synergistic cytotoxicity both in vitro and in vivo. Therefore, we propose that combining osalmid and bafilomycin A1(BafA1) may have clinical benefits against MM.

UNASSIGNED: The skin barrier is the first line of defense of the body, while skin lipids play an important role in the skin permeability barrier. Lamellar bodies are also involved in maintaining the stability of the skin permeability barrier. However, the exact origin of lamellar bodies remains unclear. Recent studies have suggested that autophagy may participate in the formation of lamellar bodies.
UNASSIGNED: This study aimed to investigate the role of autophagy in the formation of lamellar bodies in keratinocytes and the regulation of keratinocyte lipids.
UNASSIGNED: Keratinocytes were incubated with autophagy inducer Rapamycin and autophagy inhibitor Bafilomycin A1. The changes in autophagy flux were detected by Western blot, and the formation of lamellar bodies was observed by transmission electron microscopy. Furthermore, the changes in keratinocytes lipidomics were detected by liquid chromatography-mass spectrometry.
UNASSIGNED: Our research showed that the autophagy inducer promoted autophagy activation and formation of lamellar bodies in keratinocytes, while the inhibitor inhibited autophagy signals and the formation of lamellar bodies in keratinocytes. In addition, the lipidomics results revealed a significant change in glycerophospholipids after autophagy induction and autophagy inhibition.
UNASSIGNED: These results demonstrate that autophagy may play an essential role in skin lipids via glycerophospholipids pathway.

Coronavirus disease 2019 is a global pandemic caused by SARS-CoV-2. The emergence of its variant strains has posed a considerable challenge to clinical treatment. Therefore, drugs capable of inhibiting SARS-CoV-2 infection, regardless of virus variations, are in urgently need. Our results showed that the endosomal acidification inhibitor, Bafilomycin A1 (Baf-A1), had an inhibitory effect on the viral RNA synthesis of SARS-CoV-2, and its Beta and Delta variants at the concentration of 500 nM. Moreover, the human lung xenograft mouse model was used to investigate the anti-SARS-CoV-2 effect of Baf-A1. It was found that Baf-A1 significantly inhibited SARS-CoV-2 replication in the human lung xenografts by in situ hybridization and RT-PCR assays. Histopathological examination showed that Baf-A1 alleviated SARS-CoV-2-induced diffuse inflammatory infiltration of granulocytes and macrophages and alveolar endothelial cell death in human lung xenografts. In addition, immunohistochemistry analysis indicated that Baf-A1 decreased inflammatory exudation and infiltration in SARS-CoV-2-infected human lung xenografts. Therefore, Baf-A1 may be a candidate drug for SARS-CoV-2 treatment.

Activated Cdc42-associated kinase 1 (ACK1) is a promising druggable target for cancer, but its inhibitors only showed moderate effects in clinical trials. The study aimed to investigate the underlying mechanisms and improve the antitumor efficacy of ACK1 inhibitors.
RNA-seq was performed to determine the downstream pathways of ACK. Using Lasso Cox regression analysis, we built a risk signature with ACK1-related autophagy genes in the lung adenocarcinoma (LUAD) patients from The Cancer Genome Atlas (TCGA) project. The performance of the signature in predicting the tumor immune environment and response to immunotherapy and chemotherapy were assessed in LUAD. CCK8, mRFP-GFP-LC3 assay, western blot, colony formation, wound healing, and transwell migration assays were conducted to evaluate the effects of the ACK1 inhibitor on lung cancer cells. A subcutaneous NSCLC xenograft model was used for in vivo study.
RNA-seq revealed the regulatory role of ACK1 in autophagy. Furthermore, the risk signature separated LUAD patients into low- and high-risk groups with significantly different prognoses. The two groups displayed different tumor immune environments regarding 28 immune cell subsets. The low-risk groups showed high immune scores, high CTLA4 expression levels, high immunophenoscore, and low DNA mismatch repair capacity, suggesting a better response to immunotherapy. This signature also predicted sensitivity to commonly used chemotherapy and targeted drugs. In vitro, the ACK1 inhibitors (AIM-100 and Dasatinib) appeared to trigger adaptive autophagy-like response to protect lung cancer cells from apoptosis and activated the AMPK/mTOR signaling pathway, partially explaining its moderate antitumor efficacy. However, blocking lysosomal degradation with chloroquine/Bafilamycine A1 or inhibiting AMPK signaling with compound C/shPRKAA1 enhanced the ACK1 inhibitor\'s cytotoxic effects on lung cancer cells. The efficacy of the combined therapy was also verified using a mouse xenograft model.
The resulting signature from ACK1-related autophagy genes robustly predicted survival and drug sensitivity in LUAD. The lysosomal degradation inhibition improved the therapeutic effects of the ACK1 inhibitor, suggesting a potential role for autophagy in therapy evasion.

The mechanism of silk fiber formation in silkworms, Bombyx mori, is of particular scientific interest because it is closely related to the mechanical properties of silk fibers. However, there are still substantial knowledge gaps in understanding the details of this mechanism. Studies have found a pH gradient in the silk gland of silkworms. A vacuolar-type ATPase (V-ATPase) is thought to be involved in establishing this pH gradient. Although it is reported that the pH gradient plays a role in silk fibrillogenesis, the direct relationship between V-ATPase and silk mechanical properties is unclear. Thus, this study aims to clarify this relationship. We found that V-ATPase is highly and stably expressed in the anterior silk gland (ASG) and maintains the pH gradient and the fine structure of ASG. Inhibition of V-ATPase activity increased the β-sheet content and crystallinity of silk fibers. Tensile testing showed that the mechanical properties of silk fibers improved after inhibiting V-ATPase activity. All the data suggest that V-ATPase is a key factor in regulating silk fibrillogenesis and is related to the final mechanical properties of the silk fibers. V-ATPase is a potential target for silk mechanical property improvement.

Metal copper oxide nanoparticles (nano-CuO) are under mass production and have been widely utilized in many fields including catalysis, gas sensors, semiconductor materials, etc. The broad applications of nano-CuO have increased the possibility of risk to incidental exposure to the environment, and therefore, an in-depth investigation of their effects on live cells is required. This study investigated the impact of the nano-CuO on SH-SY5Y cells, and findings showed that the ratio of LC3-II/LC3-I was significantly increased in SH-SY5Y cells when the cells were treated with nano-CuO. However, if the autophagy inhibitor Bafilomycin A1 (Baf A1) was co-treated, the ratio of LC3-II/LC3-I was further improved. These outcomes might indicate that autophagy flux was permanently elevated by adding nano-CuO. Further results found highly activated levels of long noncoding RNAs (lncRNAs) under nano-CuO treatment. The data illustrate a mechanism that nano-CuO can promote autophagy and activate lncCyt b-AS/ND5-AS/ND6-AS in SH-SY5Y cells and have critical implications for nanoparticle biomedical applications.

Coronavirus disease 2019 (COVID-19) is caused by SARS-CoV-2 and broke out as a global pandemic in late 2019. The acidic pH environment of endosomes is believed to be essential for SARS-CoV-2 to be able to enter cells and begin replication. However, the clinical use of endosomal acidification inhibitors, typically chloroquine, has been controversial with this respect.
In this study, RT-qPCR method was used to detect the SARS-CoV-2N gene to evaluate viral replication. The CCK-8 assay was also used to evaluate the cytotoxic effect of SARS-CoV-2. In situ hybridization was used to examine the distribution of the SARS-CoV-2 gene in lung tissues. Hematoxylin and eosin staining was also used to evaluate virus-associated pathological changes in lung tissues.
In this study, analysis showed that endosomal acidification inhibitors, including chloroquine, bafilomycin A1 and NH4CL, significantly reduced the viral yields of SARS-CoV-2 in Vero E6, Huh-7 and 293T-ACE2 cells. Chloroquine and bafilomycin A1 also improved the viability and proliferation of Vero E6 cells after SARS-CoV-2 infection. Moreover, in the hACE2 transgenic mice model of SARS-CoV-2 infection, chloroquine and bafilomycin A1 reduced viral replication in lung tissues and alleviated viral pneumonia with reduced inflammatory exudation and infiltration in peribronchiolar and perivascular tissues, as well as improved structures of alveolar septum and pulmonary alveoli.
Our research investigated the antiviral effects of endosomal acidification inhibitors against SARS-CoV-2 in several infection models and provides an experimental basis for further mechanistic studies and drug development.

Autophagy is a highly- evolutionarily-conserved catabolic pathway activated by various cellular stresses. Recently, non-canonical autophagy (NCA), which does not require all of the ATG proteins to form autophagosome or autophagosome-like structures, has been found in various conditions. Moreover, mounting evidence has indicated that non-canonical LC3 lipidation (NCLL) may reflect NCA. We and others have reported that niclosamide (Nic), an anti-helminthic drug approved by the Food and Drug Administration, could induce canonical autophagy via a feedback downregulation of mTOR complex 1. In this study, we found that Nic could also induce NCLL, which is independent of the ULK1 complex and Beclin 1 complex, but dependent on ubiquitin-like conjugation systems. Although bafilomycin A1 and concanamycin A, two known V-ATPase inhibitors, significantly inhibited Nic-induced NCLL, Nic-induced NCLL was demonstrated to be independent of V-ATPase. In addition, the Golgi complex and vimentin were involved in Nic-induced NCLL, which might be a platform or membrane source for Nic-induced LC3-positive structures. These results would be helpful to broaden our understanding of the working mechanisms of Nic and evaluate its pharmacological activities in diseases.

Silicon-doped materials have been widely used in bone regeneration research; however, a consensus on the safety range of silicon ions has not been reached and its toxicity mechanism remains to be further elucidated. This study aims to explore whether high level of sodium metasilicate can induce toxicity effect in human umbilical vein endothelial cells (HUVEC) and the role of autophagy and apoptosis in its toxic mechanism. HUVEC was treated with different level of high silicon and then investigated with respect to morphologic change, cell viability, immunofluorescence, the level of autophagy, and apoptosis-related protein. Moreover, bafilomycin A1 (Baf A1) was applied to detect whether autophagic flux is disrupted, and 3-methyladenine (3-MA, an autophagy inhibitor) was used to determine the relationship between autophagy and apoptosis. Results demonstrated that high-level silicon induced cell viability to decrease; LC3-II, p62, and apoptosis-related proteins were up-regulated after exposure to high-dose silicon (sodium metasilicate concentration more than 1 mM). There is no significant difference in LC3-II and p62 between Baf A1 and sodium metasilicate-exposed group. Besides, 3-MA further increased the apoptotic rate by inhibiting autophagy after high silicon exposure. Collectively, high concentration of silicon can impair autophagy and induce apoptosis in human umbilical vein endothelial cells, and autophagy may play a protective role in HUVEC apoptosis. Furthermore, silicon concentration used in HUVEC should not be more than 1 mM.

The present study was designed to investigate the effects of betulinic acid on human hepatic stellate cells in vitro and C57BL/6 mice in vivo, as well as the signaling pathways involved. In this study, we explored the effects of betulinic acid on expression of alpha smooth muscle actin and autophagy-related proteins. Betulinic acid reduced pathological damage associated with liver fibrosis, as well as serum platelet-derived growth factor and serum hydroxyproline levels. Furthermore, betulinic acid downregulated the expression of alpha smooth muscle actin and type I collagen in mouse liver and upregulated the expression of microtubule-associated protein light chain 3B and autophagy-related gene 7 at the gene and protein levels. LC3II expression was increased and alpha smooth muscle actin expression was decreased in betulinic acid-treated hepatic stellate cells. Interventions with bafilomycin A1 and mCherry-GFP-LC3 adenoviruses promoted the formation of autophagosomes in hepatic stellate cells and the development of autophagic flow. Our study found that mitogen-activated protein kinase/extracellular signal-regulated kinase may be involved in the effects of betulinic acid on liver fibrosis. The present study suggests that betulinic acid has anti-hepatic fibrosis activity by inducing autophagy and could serve as a promising new agent for treating hepatic fibrosis.