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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ABSTRACT:Nonalcoholic fatty liver disease (NAFLD) is characterized by a series of pathological changes that range from simple fatty liver to nonalcoholic steatohepatitis (NASH). The objective of this study is to describe changes in global gene expression associated with the progression of human NAFLD. This study is focused on the expression levels of genes responsible for the absorption, distribution, metabolism, and elimination (ADME) of drugs. Differential gene expression between three clinically defined pathological groups-normal, steatosis, and NASH-was analyzed. Genome-wide mRNA levels in samples of human liver tissue were assayed with Affymetrix GeneChip Human 1.0ST arrays. A total of 11,633 genes exhibited altered expression out of 33,252 genes at a 5% false discovery rate. Most gene expression changes occurred in the progression from steatosis to NASH. Principal component analysis revealed that hepatic disease status was the major determinant of differential ADME gene expression rather than age or sex of sample donors. Among the 515 drug transporters and 258 drug-metabolizing enzymes (DMEs) examined, uptake transporters but not efflux transporters or DMEs were significantly over-represented in the number of genes downregulated. These results suggest that uptake transporter genes are coordinately targeted for down-regulation at the global level during the pathological development of NASH and that these patients may have decreased drug uptake capacity. This coordinated regulation of uptake transporter genes is indicative of a hepatoprotective mechanism acting to prevent accumulation of toxic intermediates in disease-compromised hepatocytes.

The epigenetic silencing of tumor suppressor genes is a common event during carcinogenesis, and often involves aberrant DNA methylation and histone modification of gene regulatory regions, resulting in the formation of a transcriptionally repressive chromatin state. Two examples include the antimetastatic, tumor suppressor genes, desmocollin 3 (DSC3) and MASPIN, which are frequently silenced in this manner in human breast cancer. Treatment of the breast tumor cell lines MDA-MB-231 and UACC 1179 with 5-aza-2 0 -deoxycytidine (5-azaCdR) induced transcriptional reactivation of both genes in a dose-dependent manner. Importantly, DSC3 and MASPIN reactivation was closely and consistently linked with significant decreases in promoter H3 K9 di-methylation. Moreover, 5-aza-CdR treatment also resulted in global decreases in H3 K9 di-methylation, an effect that was linked to its ability to mediate dose-dependent, posttranscriptional decreases in the key enzyme responsible for this epigenetic modification, G9A. Finally, small interfering RNA (siRNA)-mediated knockdown of G9A and DNMT1 led to increased MASPIN expression in MDA-MB-231 cells, to levels that were supra-additive, verifying the importance of these enzymes in maintaining multiple layers of epigenetic repression in breast tumor cells. These results highlight an additional, complimentary mechanism of action for 5-aza-CdR in the reactivation of epigenetically silenced genes, in a manner that is independent of its effects on DNA methylation, further supporting an important role for H3 K9 methylation in the aberrant repression of tumor suppressor genes in human cancer.

Using an integrated approach of epigenomic scanning and gene expression profiling, we found aberrant methylation and epigenetic silencing of a small neighborhood of contiguous genes-the HOXA gene cluster in human breast cancer. The observed transcriptional repression was localized to f100 kb of the HOXA gene cluster and did not extend to genes located upstream or downstream of the cluster. Bisulfite sequencing, chromatin immunoprecipitation, and quantitative reverse transcription-PCR analysis confirmed that the loss of expression of the HOXA gene cluster in human breast cancer is closely linked to aberrant DNA methylation and loss of permissive histone modifications in the region. Pharmacologic manipulations showed the importance of these aberrant epigenetic changes in gene silencing and support the hypothesis that aberrant DNA methylation is dominant to histone hypoacetylation. Overall, these data suggest that inactivation of the HOXA gene cluster in breast cancer may represent a new type of genomic lesion-epigenetic microdeletion. We predict that epigenetic microdeletions are common in human cancer and that they functionally resemble genetic microdeletions but are defined by epigenetic inactivation and transcriptional silencing of a relatively small set of contiguous genes along a chromosome, and that this type of genomic lesion is metastable and reversible in a classic epigenetic fashion. (Cancer Res 2006; 66(22): 10664-70)

Nonalcoholic fatty liver disease (NAFLD) may progress from simple steatosis to severe, nonalcoholic steatohepatitis (NASH) in 7%-14% of the U.S. population through a second "hit" in the form of increased oxidative stress and inflammation. Endoplasmic reticulum (ER) stress signaling and the unfolded protein response (UPR) are triggered when high levels of lipids and misfolded proteins alter ER homeostasis creating a lipotoxic environment within NAFLD livers. The objective of this study was to determine the coordinate regulation of ER stress-associated genes in the progressive stages of human NAFLD. Human liver samples categorized as normal, steatosis, NASH (Fatty), and NASH (Not Fatty) were analyzed by individual Affymetrix GeneChip Human 1.0 ST microarrays, immunoblots, and immunohistochemistry. A gene set enrichment analysis was performed on autophagy, apoptosis, lipogenesis, and ER stress/UPR gene categories. An enrichment of downregulated genes in the ER stress-associated lipogenesis and ER stress/UPR gene categories was observed in NASH. Conversely, an enrichment of upregulated ER stress-associated genes for autophagy and apoptosis gene categories was observed in NASH. Protein expression of the adaptive liver response protein STC2 and the transcription factor X-box binding protein 1 spliced (XBP-1s) were significantly elevated among NASH samples, whereas other downstream ER stress proteins including CHOP, ATF4, and phosphorylated JNK and eIF2α were not significantly changed in disease progression. Increased nuclear accumulation of total XBP-1 protein was observed in steatosis and NASH livers. The findings reveal the presence of a coordinated, adaptive transcriptional response to hepatic ER stress in human NAFLD.

No abstract

Toll-like receptor 4 (TLR4) is the principal receptor for bacterial endotoxin recognition, and functional variants in the gene confer endotoxin-hyporesponsiveness in humans. Furthermore, there is evidence that endotoxin exposure during early life is protective against the development of atopy and asthma, although this relationship remains poorly understood. It is therefore possible that genetic variation in the TLR4 locus contributes to asthma susceptibility. In this study we characterize the genetic diversity in the TLR4 locus and test for association between the common genetic variants and asthma-related phenotypes. In a cohort of 90 ethnically diverse subjects, we resequenced the TLR4 locus and identified a total of 29 single nucleotide polymorphisms. We assessed five common polymorphisms for evidence of association with asthma in two large family-based cohorts: a heterogeneous North American cohort (589 families), and a more homogenous population from northeastern Quebec, Canada (167 families). Using the transmission-disequilibrium test, we found no evidence of association for any of the polymorphisms tested, including two functional variants. Furthermore, we found no evidence for association between the TLR4 variants and four quantitative intermediate asthma- and atopy-related phenotypes. Based on these results, we found no evidence that genetic variation in TLR4 contributes to asthma susceptibility.