In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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Cancer stem cells (CSCs) are critical for cancer progression and chemoresistance. How lipid metabolism regulates CSCs and chemoresistance remains elusive. Here, we demonstrate that JAK/STAT3 regulates lipid metabolism, which promotes breast CSCs (BCSCs) and cancer chemoresistance. Inhibiting JAK/STAT3 blocks BCSC self-renewal and expression of diverse lipid metabolic genes, including carnitine palmitoyltransferase 1B (CPT1B), which encodes the critical enzyme for fatty acid β-oxidation (FAO). Moreover, mammary-adipocyte-derived leptin upregulates STAT3-induced CPT1B expression and FAO activity in BCSCs. Human breast-cancer-derived data suggest that the STAT3-CPT1B-FAO pathway promotes cancer cell stemness and chemoresistance. Blocking FAO and/or leptin re-sensitizes them to chemotherapy and inhibits BCSCs in mouse breast tumors in vivo. We identify a critical pathway for BCSC maintenance and breast cancer chemoresistance.

Autophagy is a catabolic process by which cells remove long-lived proteins and damaged organelles for recycling. Viral infections may also induce autophagic response. Here we show that hepatitis B virus (HBV), a pathogen that chronically infects ≈350 million people globally, can enhance autophagic response in cell cultures, mouse liver, and during natural infection. This enhancement of the autophagic response is not coupled by an increase of autophagic protein degradation and is dependent on the viral X protein, which binds to and enhances the enzymatic activity of phosphatidylinositol 3-kinase class III, an enzyme critical for the initiation of autophagy. Further analysis indicates that autophagy enhances HBV DNA replication, with minimal involvement of late autophagic vacuoles in this process. Our studies thus demonstrate that a DNA virus can use autophagy to enhance its own replication and indicate the possibility of targeting the autophagic pathway for the treatment of HBV patients.autophagy | hepatitis B virus DNA replication | hepatitis B virus X protein | PI3KC3A utophagy is a catabolic process by which long-lived proteins and damaged organelles are sequestered in the cytoplasm and removed for recycling. It is important for maintaining cellular homeostasis. During autophagy, membrane crescents appear in the cytoplasm. These membranes will eventually form a double-membrane structure known as autophagosomes, which will mature by fusing with lysosomes to form autolysosomes. The contents of autophagosomes will subsequently be degraded by lysosomal enzymes. Autophagy has also been implicated in innate and adaptive immune responses to the infection of microbial pathogens (1, 2). A number of viruses have been shown to induce autophagy, either completely or partially, and often with either a destructive or beneficial result to themselves. For examples, several single-stranded RNA viruses such as poliovirus, coronavirus, dengue virus, and hepatitis C virus all seem to induce the accumulation of autophagic vacuoles and use these membrane vesicles to benefit their replication (3-6). In contrast, other viruses such as herpes simplex virus-1 (HSV-1), cytomegalovirus (CMV), and Kaposi's sarcoma herpes virus (KSHV) have evolved mechanisms to suppress autophagy and, in the case of HSV-1, for its own survival (2).Hepatitis B virus (HBV) belongs to the Hepadnavirus family. This virus has a 3.2-kb circular and partially double-stranded DNA genome that contains four genes named S, C, P, and X genes. The S gene codes for the surface antigens (i.e., envelope proteins), the C gene codes for the core protein and a related protein termed precore protein, the P gene codes for the viral DNA polymerase, and the X gene codes for a multifunctional regulatory protein. After its synthesis, the core protein packages its own mRNA, which is also known as the pregenomic RNA (pgRNA), to form the core particle. The pgRNA will be converted to the DNA genome in the core particle by the viral DNA polymerase, which is also a reverse transcriptase...