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

Comparative analysis of cytoplasmic organelles in a variety of tumors relative to normal tissues generally reveals a strong diminution in mitochondrial content and in oxidative phosphorylation capacity. However, little is known about what triggers these modifications and whether or not they are physiologically reversible. We hypothesized that energy substrate availability could play an important role in this phenomenon. The physiological effects of a change in substrate availability were examined on a human cancer cell line (HeLa), focusing specifically on its ability to use glycolysis versus oxidative phosphorylation, and the effect that energy substrate type has on mitochondrial composition, structure, and function. Changes in oxidative phosphorylation were measured in vivo by a variety of techniques, including the use of two novel ratiometric green fluorescent protein biosensors, the expression level of oxidative phosphorylation and some glycolytic enzymes were determined by Western blot, mitochondrial DNA content was measured by real-time PCR, and mitochondrial morphology was monitored by both confocal and electron microscopy. Our data show that the defective mitochondrial system described in cancer cells can be dramatically improved by solely changing substrate availability and that HeLa cells can adapt their mitochondrial network structurally and functionally to derive energy by glutaminolysis only. This could also provide an explanation for the enhancement of oxidative phosphorylation capacity observed after tumor regression or removal. Our work demonstrates that the pleomorphic, highly dynamic structure of the mitochondrion can be remodeled to accommodate a change in oxidative phosphorylation activity. We compared our finding on HeLa cells with those for nontransformed fibroblasts to help distinguish the regulatory pathways.

Surgical meshes, in particular those used to repair hernias, have been in use since 1891. Since then, research in the area has expanded, given the vast number of post-surgery complications such as infection, fibrosis, adhesions, mesh rejection, and hernia recurrence. Researchers have focused on the analysis and implementation of a wide range of materials: meshes with different fiber size and porosity, a variety of manufacturing methods, and certainly a variety of surgical and implantation procedures. Currently, surface modification methods and development of nanofiber based systems are actively being explored as areas of opportunity to retain material strength and increase biocompatibility of available meshes. This review summarizes the history of surgical meshes and presents an overview of commercial surgical meshes, their properties, manufacturing methods, and observed biological response, as well as the requirements for an ideal surgical mesh and potential manufacturing methods.

The inner membrane system of mitochondria is known to consist of two contiguous but distinct membranes: the inner boundary membrane, which apposes the outer membrane, and the cristal membrane, which forms tubules or lamellae in the interior. Using immunolabeling and transmission electron microscopy of bovine heart tissue, we have calculated that around 94% of both Complex III of the respiratory chain and the ATP synthase are located in the cristal membrane, and only around 6% of either is in the inner boundary membrane. When accounting for the topographical ratio of cristal membrane versus inner boundary membrane, we ¢nd that both complexes exist at a 2.2^2.6-fold higher concentration in the cristal membrane. The residual protein in the inner boundary membrane may be newly assembled complexes destined for cristal membranes. Our results argue for restricted di¡usion of complexes through the cristal junctions and indicate that the mitochondrial cristae comprise a regulated submitochondrial compartment specialized for ATP production.

Mitochondrial DNA (mtDNA) is packaged into DNA-protein assemblies called nucleoids, but the mode of mtDNA propagation via the nucleoid remains controversial. Two mechanisms have been proposed: nucleoids may consistently maintain their mtDNA content faithfully, or nucleoids may exchange mtDNAs dynamically. To test these models directly, two cell lines were fused, each homoplasmic for a partially deleted mtDNA in which the deletions were nonoverlapping and each deficient in mitochondrial protein synthesis, thus allowing the first unequivocal visualization of two mtDNAs at the nucleoid level. The two mtDNAs transcomplemented to restore mitochondrial protein synthesis but were consistently maintained in discrete nucleoids that did not intermix stably. These results indicate that mitochondrial nucleoids tightly regulate their genetic content rather than freely exchanging mtDNAs. This genetic autonomy provides a molecular mechanism to explain patterns of mitochondrial genetic inheritance, in addition to facilitating therapeutic methods to eliminate deleterious mtDNA mutations.