Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

A late Archaean radiating dyke swarm as possible clue to the origin of the Bushveld Complex

The Bushveld Complex in South Africa represents the world's largest intrusion of magnesium-and iron-rich magmas. The Bushveld magmas were emplaced beneath the Transvaal basin(1) similar to 2.06 billion years ago(2,3), but their origin remains elusive. The magmas may have formed in response to an upwelling mantle plume(4), ancient subduction5 or melting triggered by a meteorite impact(6). Here we use U-Pb dating of baddeleyite crystals to date a series of mafic magmatic dykes located east of the Transvaal basin. We find that these dykes formed between 2.70 and 2.66 billion years ago, roughly 600 million years before the Bushveld magmas were emplaced. Collectively, the geometry of the dykes forms a radiating swarm converging towards a focal point in the eastern part of the Bushveld Complex. Such radiating swarms typically record the impact of a mantle plume head that injected large volumes of magma into the crust and at the base of the lithosphere. We propose that subsequent cooling and metamorphism of these mantle-plume-derived rocks caused them to increase in density and sink, triggering subsidence of the Transvaal basin. The dense rocks may later have sunk away into the mantle, with the delamination causing the inflow of hot mantle that initiated production of the voluminous Bushveld magmas about 600 million years after the mantle plume impact

Peripheral Corticotropin-Releasing Factor Receptor Type 2 Activation Increases Colonic Blood Flow Through Nitric Oxide Pathway in Rats

BACKGROUND: Corticotropin-releasing factor (CRF) peptides exert profound effects on the secretomotor function of the gastrointestinal tract. Nevertheless, despite the presence of CRF peptides and receptors in colonic tissue, their influence on colonic blood flow (CBF) is unknown. AIM: To determine the effect and mechanism of members of the CRF peptide family on CBF in isoflurane-anesthetized rats. METHODS: Proximal CBF was measured with laser Doppler flowmetry simultaneously with mean arterial blood pressure (MABP) measurement. Rats were injected with intravenous human/rat CRF (CRF(1)>CRF(2) affinity), mouse urocortin 2 (mUcn2, selective CRF(2) agonist) or sauvagine (SVG, CRF(2)>CRF(1) affinity) at 1 – 30 μg/kg. The nitric oxide (NO) synthase inhibitor, L-NAME (3 mg/kg, iv), the cyclooxygenase inhibitor, indomethacin (Indo, 5 mg/kg, ip) or selective CRF(2) antagonist, astressin(2)-B (Ast(2)B, 50 μg/kg, iv) was given before SVG injection (10 μg/kg, iv). RESULTS: SVG and mUcn2 dose-dependently increased CBF while decreasing MABP and colonic vascular resistance (CVR). CRF had no effect on CBF, but increased CVR. The hyperemic effect of SVG was inhibited by L-NAME but not by Indo, whereas hypotension was partially reduced by L-NAME. Sensory denervation had no effect on SVG-induced changes. Ast(2)B inhibited SVG-induced hyperemia and decreased CVR, and partially reduced the hypotension. CONCLUSIONS: Peripheral CRF(2) activation induces colonic hyperemia through NO synthesis, without involving prostaglandin synthesis or sensory nerve activation, suggesting a direct action on the endothelium and myenteric neurons. Members of the CRF peptide family may protect the colonic mucosal via the activation of the CRF(2) receptor