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.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

BACKGROUND\nAlthough many patients with intermediate-grade or high-grade (aggressive) non-Hodgkin's lymphoma are cured by combination chemotherapy, the remainder are not cured and ultimately die of their disease. The Ann Arbor classification, used to determine the stage of this disease, does not consistently distinguish between patients with different long-term prognoses. This project was undertaken to develop a model for predicting outcome in patients with aggressive non-Hodgkin's lymphoma on the basis of the patients' clinical characteristics before treatment.\n\n\nMETHODS\nAdults with aggressive non-Hodgkin's lymphoma from 16 institutions and cooperative groups in the United States, Europe, and Canada who were treated between 1982 and 1987 with combination-chemotherapy regimens containing doxorubicin were evaluated for clinical features predictive of overall survival and relapse-free survival. Features that remained independently significant in step-down regression analyses of survival were incorporated into models that identified groups of patients of all ages and groups of patients no more than 60 years old with different risks of death.\n\n\nRESULTS\nIn 2031 patients of all ages, our model, based on age, tumor stage, serum lactate dehydrogenase concentration, performance status, and number of extranodal disease sites, identified four risk groups with predicted five-year survival rates of 73 percent, 51 percent, 43 percent, and 26 percent. In 1274 patients 60 or younger, an age-adjusted model based on tumor stage, lactate dehydrogenase level, and performance status identified four risk groups with predicted five-year survival rates of 83 percent, 69 percent, 46 percent, and 32 percent. In both models, the increased risk of death was due to both a lower rate of complete responses and a higher rate of relapse from complete response. These two indexes, called the international index and the age-adjusted international index, were significantly more accurate than the Ann Arbor classification in predicting long-term survival.\n\n\nCONCLUSIONS\nThe international index and the age-adjusted international index should be used in the design of future therapeutic trials in patients with aggressive non-Hodgkin's lymphoma and in the selection of appropriate therapeutic approaches for individual patients.

A predictive model for aggressive non-Hodgkin's lymphoma

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

https://biblio.ugent.be/publication/8555786/file/8555788.pdf

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

An understanding of the basic defect in the inherited disorder cystic fibrosis requires cloning of the cystic fibrosis gene and definition of its protein product. In the absence of direct functional information, chromosomal map position is a guide for locating the gene. Chromosome walking and jumping and complementary DNA hybridization were used to isolate DNA sequences, encompassing more than 500,000 base pairs, from the cystic fibrosis region on the long arm of human chromosome 7. Several transcribed sequences and conserved segments were identified in this cloned region. One of these corresponds to the cystic fibrosis gene and spans approximately 250,000 base pairs of genomic DNA.

https://science.sciencemag.org/content/sci/245/4922/1059.full.pdf

Identification of the cystic fibrosis gene: Chromosome walking and jumping

The heparan sulfate on the surface of all adherent cells modulates the actions of a large number of extracellular ligands. Members of both cell surface heparan sulfate proteoglycan families, the transmembrane syndecans and the glycosylphosphoinositide-linked glypicans, bind these ligands and enhance formation of their receptor-signaling complexes. These heparan sulfate proteoglycans also immobilize and regulate the turnover of ligands that act at the cell surface. The extracellular domains of these proteoglycans can be shed from the cell surface, generating soluble heparan sulfate proteoglycans that can inhibit interactions at the cell surface. Recent analyses of genetic defects in Drosophila melanogaster, mice, and humans confirm most of these activities in vivo and identify additional processes that involve cell surface heparan sulfate proteoglycans. This chapter focuses on the mechanisms underlying these activities and on the cellular functions that they regulate.

Functions of Cell Surface Heparan Sulfate Proteoglycans

Following ingestion of bovine serum albumin (BSA), human infants and rabbits synthesize circulating anti-BSA. Using a similar protocol, the serum antibody response was examined in C3H mice offered 0.1, 1.0 or 6.0% BSA for 10 or 56 days. Sera from these animals contained similar amounts of antigen, but anti-BSA was undetectable. Half the animals in each group were subsequently injected i.v. with 50.0 mg BSA. Mice fed 1.0 or 6.0% BSA were unresponsive to this challenge, but animals fed 0.1% BSA responded comparably to i.v. immunized, non-fed controls. The remaining animals in each group were injected s.c. with 1.0 mg BSA in adjuvant. Most of these mice produced circulating anti-BSA. However, these responses were significantly reduced relative to s.c. immunized, non-fed controls. Hyporesponsiveness again was least after 0.1% BSA ingestion. CF1 mice and BN, HCR and HCS rats fed 0.1% BSA for 56 days also did not produce circulating antibodies during ingestion, and almost all were hyporesponsive after parenteral challenge. Suppression of specific antibody responses also was observed in C3H mice fed and challenged with diphtheria toxoid. The data suggest that under identical experimental conditions, ingestion of soluble protein antigens induces a state of systemic hyporesponsiveness in mice and rats in contrast to the systemic immunity demonstrated in human neonates and adult rabbits.

Systemic antibody responses of different species following ingestion of soluble protein antigens.

We have recently developed a method to detect tumor-specific rearrangement of the IgH gene in interphase nuclei by fluorescence in situ hybridization. Tumor-specific IgH gene rearrangement is equivalent to 14q32.33 translocation. Using this approach, we detected 14q32.33 translocation in 29 of 70 patients with B-cell non-Hodgkin's lymphoma (NHL). Chromosome t(3;14) was found in 10 of these 29 patients, and were demonstrated as a fusion signal of BCL6 and VH gene probes in interphase nuclei. Furthermore, in another series of 11 patients and a NHL cell line, we demonstrated t(14;18) and t(11;14) in interphase and metaphase cells with a combination of BCL2 (or PRADI) with IgH gene probes. Interphase FISH with lymphoma-associated gene probes is a rapid procedure for cytogenetic diagnosis of B-cell NHL.

Rapid detection of lymphoma-specific translocations in interphase nuclei of non-Hodgkin's lymphoma by fluorescence in situ hybridization.

RNA interference (RNAi) is a process in which double-stranded RNA (dsRNA) molecules mediate the inhibition of gene expression. RNAi in C. elegans can be achieved by simply feeding animals with bacteria expressing dsRNA against the gene of interest. This "feeding" method has made it possible to conduct genome-wide RNAi experiments for the systematic knockdown and subsequent investigation of almost every single gene in the genome. Historically, these genome-scale RNAi screens have been labor and time intensive. However, recent advances in automated, high-throughput methodologies have allowed the development of more rapid and efficient screening protocols. In this report, we describe a fast and efficient, liquid-based method for genome-wide RNAi screening.

High-Throughput, Liquid-Based Genome-Wide RNAi Screening in C. elegans.

Abstract Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans ( C. elegans ) null for a cysteine protease inhibitor, srp-6 , undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3 , respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans , this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors. 

/pdf/lysoptosis-is-an-evolutionarily-conserved-cell-death-pathway-2oy3gytz.pdf

Gary A. Silverman

Papers

Systemic antibody responses of different species following ingestion of soluble protein antigens.

Rapid detection of lymphoma-specific translocations in interphase nuclei of non-Hodgkin's lymphoma by fluorescence in situ hybridization.

High-Throughput, Liquid-Based Genome-Wide RNAi Screening in C. elegans.

Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins

the RNAi pathway to misfolded protein turnover