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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(25D 3 ) 3 (2). The second step is conventionally known to take place in the kidneys, but increasing number of tissues have been found to express 1␣-hydroxylase (Cyp27B1), the enzyme responsible for the final and rate-limiting step in the synthesis of the active 1,25-dihydroxyvitamin D 3 (1,25D 3 ) (3, 4). Expression of 1␣-hydroxylase has been reported in epithelial cells of the skin (keratinocytes) (5, 6), intestine (7, 8), breast (9), and prostate (10) and in cells of the immune system including macrophages (11-13), monocytes VDR modulates the expression of a long list of genes in a celland tissue-specific manner. A number of these genes play a role in immunity. The first evidence suggesting that vitamin D has important immunomodulatory effects comes from epidemiological data showing that individuals with low 25D 3 levels are more susceptible to Mycbacterium tuberculosis infection and often have a more severe course of disease (20,21). Furthermore, several casecontrol studies have found an association between VDR polymorphisms and susceptibility to tuberculosis (22)(23)(24). A recent translational study indicates that the reason for these findings is insufficient levels of the antimicrobial protein, cathelicidin, in individuals with low vitamin D levels. In this study, activation of TLR2/1 by a mycobacterial ligand triggered up-regulation of cathelicidin mRNA and increased killing of mycobacteria by macrophages in the presence of vitamin D (25). A subsequent study in skin epithelial cells (keratinocytes) showed that skin injury or infection (TLR2/6 ligand) leads to activation of vitamin D and upregulation of at least two vitamin D-dependent genes, the cathelicidin antimicrobial peptide and the TLR coreceptor, CD14, involved in innate immunity (6). The genes encoding for cathelicidin (26 -28) and CD14 (29) have VDREs within their promoters and are positively regulated by vitamin D. Both are primarily expressed in myeloid cells but have also been found in respiratory

Endotoxin-induced cytokine gene transcription in monocytes and macrophages is regulated in part by NF-B. We have previously shown that the p38 mitogenactivated protein (MAP) kinase is necessary for endotoxin-induced cytokine gene transcription. Due to the fact that most cytokine promoter sequences have active NF-B sites, we hypothesized that the p38 MAP kinase was necessary for NF-B-dependent gene expression. We found that NF-B-dependent gene expression was reduced to near control levels with either SB 203580 or a dominant-negative p38 MAP kinase expression vector. Inhibition of the p38 MAP kinase did not alter NF-B activation at any level, but it significantly reduced the DNA binding of TATA-binding protein (TBP) to the TATA box. The dominant-negative p38 MAP kinase expression vector interfered with the direct interaction of native TFIID (TBP) with a co-transfected p65 fusion protein. Likewise, this dominant-negative plasmid also interfered with the direct interaction of a co-transfected TBP fusion protein with the native p65 subunit. The p38 kinase also phosphorylated TFIID (TBP) in vitro, and SB 203580 inhibited phosphorylation of TFIID (TBP) in vivo. Thus, the p38 MAP kinase regulates NF-B-dependent gene transcription, in part, by modulating activation of TFIID (TBP). Cytokine gene expression in endotoxin (LPS)1 -stimulated macrophages is regulated, at least in part, at the level of transcription. A transcription factor that is necessary for the transcription of many cytokine genes is nuclear factor B (NF-B) (1-4). In addition to others, we have previously shown that NF-B binds to specific cytokine promoter sequences (1-8). NF-B is composed of heterodimers (most commonly p50 and p65) of members of the Rel family of transcription factors. In quiescent cells the heterodimers are kept in the cytoplasm by an inhibitor protein, IB (9 -11). NF-B translocation and DNA binding is dependent on IB kinase (IKK), which phosphorylates IB on serines within the amino-terminal domain (12)(13)(14)(15)(16)(17). This phosphorylation results in IB degradation, thus allowing NF-B translocation to the nucleus. Other factors, however, have been shown to be essential for NF-B transcriptional activation, especially phosphorylation of the p65 subunit of NF-B in one of two of its transactivation domains (18). In addition, the association of the carboxyl terminus of p65 with basal transcription factors, such as transcription factor IIB (TFIIB) and TATA-binding protein (TBP), is known to be important for transcriptional regulation of .One family of kinases that is essential for transferring signals from the cell surface to the nucleus is the mitogen-activated protein (MAP) kinases. We and others have shown that the p38 MAP kinase is critical for LPS-induced cytokine gene expression (23)(24)(25)(26).Some of these studies showed that LPS, interleukin 1, and osmotic stress activate p38 MAP kinases, and inhibition with SB 203580, a competitive inhibitor of the p38 MAP kinases, reduced cytokine release but did not affect cytokine mRNA accumu...

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Smoking is associated with a wide variety of adverse health outcomes including cancer, chronic obstructive pulmonary disease, diabetes, depression and heart disease. Unfortunately, the molecular mechanisms through which these effects are conveyed are not clearly understood. To examine the potential role of epigenetic factors in these processes, we examined the relationship of smoking to genome wide methylation and gene expression using biomaterial from two independent samples, lymphoblast DNA and RNA (n=119) and lung alveolar macrophage DNA (n=19). We found that in both samples current smoking status was associated with significant changes in DNA methylation, in particular at the aryl hydrocarbon receptor repressor (AHRR), a known tumor suppressor. Both baseline DNA methylation and smoker associated DNA methylation signatures at AHRR were highly correlated (r=0.94 and 0.45, respectively). DNA methylation at the most differentially methylated AHRR CpG residue in both samples, cg0557592, was significantly associated with AHRR gene expression. Pathway analysis of lymphoblast data (genes with most significant methylation changes) demonstrated enrichment in protein kinase C pathways and in TGF beta signaling pathways. For alveolar macrophages, pathway analysis demonstrated alterations in inflammation-related processes. We conclude that smoking is associated with functionally significant genome wide changes in DNA methylation in both lymphoblasts and pulmonary macrophages and that further integrated investigations of these epigenetic effects of smoking on carcinogenesis and other related co-morbidities are indicated.

BackgroundRegular smoking is associated with a wide variety of syndromes with prominent inflammatory components such as cancer, obesity and type 2 diabetes. Heavy regular smoking is also associated with changes in the DNA methylation of peripheral mononuclear cells. However, in younger smokers, inflammatory epigenetic findings are largely absent which suggests the inflammatory response(s) to smoking may be dose dependent. To help understand whether peripheral mononuclear cells have a role in mediating these responses in older smokers with higher cumulative smoke exposure, we examined genome-wide DNA methylation in a group of well characterized adult African American subjects informative for smoking, as well as serum C-reactive protein (CRP) and interleukin-6 receptor (IL6R) levels. In addition, complementary bioinformatic analyses were conducted to delineate possible pathways affected by long-term smoking.ResultsGenome-wide DNA methylation analysis with respect to smoking status yielded 910 significant loci after Benjamini-Hochberg correction. In particular, two loci from the AHRR gene (cg05575921 and cg23576855) and one locus from the GPR15 gene (cg19859270) were identified as highly significantly differentially methylated between smokers and non-smokers. The bioinformatic analyses showed that long-term chronic smoking is associated with altered promoter DNA methylation of genes coding for proteins mapping to critical sub-networks moderating inflammation, immune function, and coagulation.ConclusionsWe conclude that chronic regular smoking is associated with changes in peripheral mononuclear cell methylation signature which perturb inflammatory and immune function pathways and may contribute to increased vulnerability for complex illnesses with inflammatory components.

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States, and cigarette smoking is the major risk factor for COPD. Fibroblasts play an important role in repair and lung homeostasis. Recent studies have demonstrated a reduced growth rate for lung fibroblasts in patients with COPD. In this study we examined the effect of cigarette smoke extract (CSE) on fibroblast proliferative capacity. We found that cigarette smoke stopped proliferation of lung fibroblasts and upregulated two pathways linked to cell senescence (a biological process associated with cell longevity and an inability to replicate), p53 and p16-retinoblastoma protein pathways. We compared a single exposure of CSE to multiple exposures over an extended time course. A single exposure to CSE led to cell growth inhibition at multiple phases of the cell cycle without killing the cells. The decrease in proliferation was accompanied by increased ATM, p53, and p21 activity. However, several important senescent markers were not present in the cells at an earlier time point. When we examined multiple exposures to CSE, we found that the cells had profound growth arrest, a flat and enlarged morphology, upregulated p16, and senescenceassociated ␤-galactosidase activity, which is consistent with a classic senescent phenotype. These observations suggest that while a single exposure to cigarette smoke inhibits normal fibroblast proliferation (required for lung repair), multiple exposures to cigarette smoke move cells into an irreversible state of senescence. This inability to repair lung injury may be an essential feature of emphysema.