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

Programmed cell death is an active process of self destruction that is important in both the development and maintenance of multicellular animals. The molecular mechanisms controlling activation or suppression of programmed cell death are largely unknown. Apoptosis, a morphologically and biochemically defined type of programmed cell death commonly seen in vertebrates, was found to be initiated during baculovirus replication in insect cells. A specific viral gene product, p35, was identified as being responsible for blocking the apoptotic response. Identification of the function of this gene will allow further definition of the molecular pathways involved in the regulation of programmed cell death and may identify the role of apoptosis in invertebrate viral defense systems.

Scrape loading and sonication loading are two recently described methods of introducing macromolecules into living cells. We have tested the efficacy of these methods for transfection ofmammalian cells with exogenous DNA, using selection systems based either on resistance to the drug G418

Assessment of undergraduate research (UR) programs using participant surveys has produced a wealth of information about design, implementation, and perceived benefits of UR programs. However, measurement of student participation university wide, and the potential contribution of research experience to student success, also require the study of extrinsic measures. In this essay, institutional data on student credit-hour generation and grade point average (GPA) from the University of Georgia are used to approach these questions. Institutional data provide a measure of annual enrollment in UR classes in diverse disciplines. This operational definition allows accurate and retrospective analysis, but does not measure all modes of engagement in UR. Cumulative GPA is proposed as a quantitative extrinsic measure of student success. Initial results show that extended participation in research for more than a single semester is correlated with an increase in GPA, even after using SAT to control for the initial ability level of the students. While the authors acknowledge that correlation does not prove causality, continued efforts to measure the impact of UR programs on student outcomes using GPA or an alternate extrinsic measure is needed for development of evidence-based programmatic recommendations.

Abstract. The interaction with actin and intracellular localization of the 30,000-D actin-binding protein from the cellular slime mold Dictyostelium discoideum have been investigated to analyze the potential contributions of this protein to cell structure and movement. The formation of anisotropic cross-linked filament networks (bundles) containing actin and the 30,000-D protein has been observed by electron microscopy, light scattering, viscometry, and polarization microscopy. Cosedimentation experiments indicate that a maximum of one molecule of the 30,000-D protein can bind to 10 actin monomers in filaments with an apparent association constant of 1 x 107 liters/mol. Inhibition of the interaction of the 30,000-D protein with actin by either magnesium or calcium was observed by viscometry, light scattering, polarization microscopy, and direct binding assays. However, the concentration of magnesium required to diminish the interaction is >100 times greater than that of calcium. The association constant of the 30,000-D protein for actin is 4.2 x 106 liters/mol, or <1 x ltY liters/mol in the presence of increased concentrations of either Mg 2÷ or Ca 2÷, respectively. Enzyme-linked immunoassays indicate that the 30,000-D protein comprises 0.04 % of the protein in D. discoideum. Extensive interaction of the 30,000-D protein with actin in cytoplasm is predicted from these measurements of the concentration of this protein and its affinity for actin.The distribution of the 30,000-D protein was analyzed by immunofluorescence microscopy using monospecific affinity-purified polyclonal antibody. The 30,000-D protein exhibits a diffuse distribution in cytoplasm, is excluded from prominent organeUes, and is quite prominent in fine extensions protruding from the cell surface. The number, length, and distribution of these extensions containing the 30,000-D protein are similar to those of filopodia observed by scanning electron microscopy. To analyze the effects of cell thickness and the distribution of organelles on the immunofluorescence localization, fluorescein-labeled BSA was incorporated into the cytoplasm of living cells before fixation and staining using a sonication loading technique. The results indicate that the 30,000-D protein is selectively incorporated into filopodia. These results provide a clear distinction between the multiple actin-cross-linking proteins present in D. discoideum, and suggest that the 30,000-D protein contributes to organization of bundles of actin filaments in filopodia.