Guidelines for the use and interpretation of assays for monitoring autophagy

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.

A coronary heart disease risk model for predicting the effect of potent antiretroviral therapy in HIV-1 infected men

BACKGROUND: Many HIV-infected patients on highly active antiretroviral therapy (HAART) experience metabolic complications including dyslipidaemia and insulin resistance, which may increase their coronary heart disease (CHD) risk. We developed a prognostic model for CHD tailored to the changes in risk factors observed in patients starting HAART. METHODS: Data from five cohort studies (British Regional Heart Study, Caerphilly and Speedwell Studies, Framingham Offspring Study, Whitehall II) on 13,100 men aged 40-70 and 114,443 years of follow up were used. CHD was defined as myocardial infarction or death from CHD. Model fit was assessed using the Akaike Information Criterion; generalizability across cohorts was examined using internal-external cross-validation. RESULTS: A parametric model based on the Gompertz distribution generalized best. Variables included in the model were systolic blood pressure, total cholesterol, high-density lipoprotein cholesterol, triglyceride, glucose, diabetes mellitus, body mass index and smoking status. Compared with patients not on HAART, the estimated CHD hazard ratio (HR) for patients on HAART was 1.46 (95% CI 1.15-1.86) for moderate and 2.48 (95% CI 1.76-3.51) for severe metabolic complications. CONCLUSIONS: The change in the risk of CHD in HIV-infected men starting HAART can be estimated based on typical changes in risk factors, assuming that HRs estimated using data from non-infected men are applicable to HIV-infected men. Based on this model the risk of CHD is likely to increase, but increases may often be modest, and could be offset by lifestyle changes.

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.

Sēfer lēv ṭôv : we-nôsāf ʿalāw liqqûṭîm ḥadāšîm ...

[Isaak Ben-Eljakim aus Posen]

Sēfer Maʿaśe H' : do einn werṭ der tzilṭ wi H' hoṭ giṭon gruśi nisim durch di talmide ḥachomim

[Simon Akiba Bär Ben-Josef]

Tseʾenah u-reʾenah : ṿe-hu ḥamishah ḥumshe Torah ṿe-haftarot le-khol ha-shanah ṿe-ḥamesh megilot ṿe-ha-targumi' bi-l. A. [bi-leshon Ashkenaz]

[Yaʿaḳov ben Yitsḥaḳ Askhenazi]

Sēfer Maʿaśe H' : ... maʿaśîm gedôlîm we-niflā'îm ... bi-lešôn haq-qôdeš û-vi-lešôn aškenaz

[Simon Akiba Bär Ben-Josef]

Sēfer Maʿaśe H' : do einn werṭ der tzilṭ wi H' huṭ gruśi nisim giṭon durch talmide ḥachomim ...

[Simon Akiba Bär Ben-Josef]

Sēfer Maʿaśe H' : do inn werṭ der tzilṭ wi H' J' hoṭ giṭon grośi nisim durch di talmide ḥachomim

[Simon Akiba Bär Ben-Josef]

[Sefer Yetsirah] id est Liber Iezirah, qui Abrahamo patriarchæ adscribitur : unà cum commentario rabi Abraham f. D super 32 Semitis Sapientiæ, à quibus liber Iezirah incipit

translatus & notis illustratus à Joanne Stephano Rittanglio

Sedimentology and Lead and Strontium isotope ratios of Miocene sediments from the Kerguelen Plateau

Characterization of sediment from Ocean Drilling Program Site 745, representing the East Kerguelen Ridge sediment drift, addresses important issues surrounding the timing of Miocene to present East Antarctic ice sheet stability and oceanic environmental change. Our results show three periods of greatly enhanced accumulation of Antarctic-derived sediment, at 6.4-5.9 Ma, 4.9-4.4 Ma and 1.1-0.8 Ma, potentially indicative of warmer, less stable ice sheets at these times. Conversely, the accumulation of Antarctic-derived material is comparatively less during the middle of the Pliocene warm epoch (4.8-3.2 Ma). The deep flow forming the Kerguelen drift was stronger during the latest Miocene and earliest Pliocene and has decreased in intensity continuously since then.

Age data of ODP Site 181-1124

ODP Site 1124, located 600 km east of the North Island of New Zealand, records post-middle Oligocene variations in the Pacific Deep Western Boundary Current (DWBC) and New Zealand's climatic and tectonic evolution. Sediment parameters, such as terrigenous grain size, flux, magnetic fabric, and non-depositional episodes, are used to interpret DWBC intensity and Antarctic climate. Interpretations of DWBC velocities indicate that the Antarctic Circumpolar Current reached modern intensities at ~23 Ma, as the tectonic seaways expanded, completing the thermal isolation of Antarctica. Periods of more intense bottom water formation are suggested by the presence of hiatuses formed under the DWBC at 22.5-17.6, 16.5-15, and 14-11 Ma. The oldest interval of high current intensity occurs within a climatically warm period during which the intensity of thermohaline circulation around Antarctica increased as a result of recent opening of circum-Antarctic gateways. The younger hiatuses represent glacial periods on Antarctica and major fluctuations in the East Antarctic Ice Sheet, whereas intervals around the hiatuses represent times of relative warmth, but with continued current activity. The period between 11 to 9 Ma is characterized by conditions surrounding a high velocity DWBC around the time of the formation and stabilization of the West Antarctic Ice Sheet. The increased terrigenous input may result from either changing Antarctic conditions or more direct sediment transport from New Zealand. The Pacific DWBC did not exert a major influence on sedimentation at Site 1124 from 9 Ma to the present; the late Miocene to Pleistocene sequence is more influenced by the climatic and tectonic history of New Zealand. Despite the apparent potential for increased sediment supply to this site from changes in sediment channeling, increasing rates of mountain uplift, and volcanic activity, terrigenous fluxes remain low and constant throughout this younger period.

Grain size and magnetic fabric analyses

The combined use of grain size and magnetic fabric analyses provides the ability to discriminate among depositional environments in deep-sea terrigenous sediments. We analyzed samples from three different depositional settings: turbidites, pelagic or hemipelagic interlayers, and sediment drifts. Results indicate that sediment samples from these different environments can be distinguished from each other on the basis of their median grain size, sorting, as well as the intensity and shape of magnetic fabric as determined from an examination of the anisotropy of magnetic susceptibility. We use these discriminators to interpret downcore samples from the Bermuda Rise sediment drift. We find that the finer grains of the Bermuda Rise (relative to the Blake Outer Ridge) do not result from lower depositional energy (current speed) and so may reflect a difference in the nature of sediment being delivered to the site (i.e., distance from source) between the two locations.