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.

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

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

Multilingualism, Legal Drafting and Interpretation of Bilingual Law in Italy

The article begins with a short history of the current Italian language, as an example of a dialect evolving and becoming elevated to the status of a national language. Next, an overview of Italy as characterized by multilingualism and of the different minority languages is offered. A third part is devoted to the different legal languages of Italian law and particularly to the consequences of multilingualism in Italy, which refers to the obligation to draft some local laws in two or tree languages. Multilingual drafting concerns institutions – and therefore concepts – of Italian law which are applied within one single legal system, namely the Italian one, and are merely expressed in a legal language which is not only Italian, but German, French or Ladin. This part is discussed more in deep. The article underlines that legal multilingualism in Italy is a rather unexplored research field. As in Europe there is a clear need for studies inquiring the problem of intepretation and application of mulitlingual law, the praxis and the operative reality of the “regional” legal languages in Italy would probably deserve more attention.

Patterns and Correlates of Expressed Emotion, Perceived Criticism, and Rearing Style in First Admitted Early-Onset Schizophrenia Spectrum Disorders

The aim of this study was to assess patterns and correlates of family variables in 31 adolescents treated for their first episode of a schizophrenia spectrum disorder (early-onset schizophrenia [EOS]). Expressed emotion, perceived criticism, and rearing style were assessed. Potential correlates were patient psychopathology, premorbid adjustment, illness duration, quality of life (QoL), sociodemographic variables, patient and caregiver "illness concept," and caregiver personality traits and support. Families were rated as critical more frequently by patients than raters (55% vs. 13%). Perceived criticism was associated with worse QoL in relationship with parents and peers. An adverse rearing style was associated with a negative illness concept in patients, particularly with less trust in their physician. Future research should examine perceived criticism as a predictor of relapse and indicator of adolescents with EOS who need extended support and treatment. Rearing style should be carefully observed because of its link with patients' illness concept and, potentially, to service engagement and medication adherence

Endovascular treatment for acute ischemic stroke patients: implications and interpretation of IMS III, MR RESCUE, and SYNTHESIS EXPANSION trials: A report from the Working Group of International Congress of Interventional Neurology

OBJECTIVE The results of Interventional Management of Stroke (IMS) III, Magnetic Resonance and REcanalization of Stroke Clots Using Embolectomy (MR RESCUE), and SYNTHESIS EXPANSION trials are expected to affect the practice of endovascular treatment for acute ischemic stroke. The purpose of this report is to review the components of the designs and methods of these trials and to describe the influence of those components on the interpretation of trial results. METHODS A critical review of trial design and conduct of IMS III, MR RESCUE, and SYNTHESIS EXPANSION is performed with emphasis on patient selection, shortcomings in procedural aspects, and methodology of data ascertainment and analysis. The influence of each component is estimated based on published literature including multicenter clinical trials reporting on endovascular treatment for acute ischemic stroke and myocardial infarction. RESULTS We critically examined the time interval between symptom onset and treatment and rates of angiographic recanalization to differentiate between "endovascular treatment" and "parameter optimized endovascular treatment" as it relates to the IMS III, MR RESCUE, and SYNTHESIS EXPANSION trials. All the three trials failed to effectively test "parameter optimized endovascular treatment" due to the delay between symptom onset and treatment and less than optimal rates of recanalization. In all the three trials, the magnitude of benefit with endovascular treatment required to reject the null hypothesis was larger than could be expected based on previous studies. The IMS III and SYNTHESIS EXPANSION trials demonstrated that rates of symptomatic intracerebral hemorrhages subsequent to treatment are similar between IV thrombolytics and endovascular treatment in matched acute ischemic stroke patients. The trials also indirectly validated the superiority/equivalence of IV thrombolytics (compared with endovascular treatment) in patients with minor neurological deficits and those without large vessel occlusion on computed tomographic/magnetic resonance angiography. CONCLUSIONS The results do not support a large magnitude benefit of endovascular treatment in subjects randomized in all the three trials. The possibility that benefits of a smaller magnitude exist in certain patient populations cannot be excluded. Large magnitude benefits can be expected with implementation of "parameter optimized endovascular treatment" in patients with ischemic stroke who are candidates for IV thrombolytics.

Recommendations for sex/gender neuroimaging research: Key principles and implications for research design, analysis and interpretation

Neuroimaging (NI) technologies are having increasing impact in the study of complex cognitive and social processes. In this emerging field of social cognitive neuroscience, a central goal should be to increase the understanding of the interaction between the neurobiology of the individual and the environment in which humans develop and function. The study of sex/gender is often a focus for NI research, and may be motivated by a desire to better understand general developmental principles, mental health problems that show female-male disparities, and gendered differences in society. In order to ensure the maximum possible contribution of NI research to these goals, we draw attention to four key principles—overlap, mosaicism, contingency and entanglement—that have emerged from sex/gender research and that should inform NI research design, analysis and interpretation. We discuss the implications of these principles in the form of constructive guidelines and suggestions for researchers, editors, reviewers and science communicators.

Mercury species concentrations near Casey station, Antarctica and along the SR3 CASO-GEOTRACES transect

We present here the first mercury speciation study in the water column of the Southern Ocean, using a high-resolution south-to-north section (27 stations from 65.50°S to 44.00°S) with up to 15 depths (0-4440 m) between Antarctica and Tasmania (Australia) along the 140°E meridian. In addition, in order to explore the role of sea ice in Hg cycling, a study of mercury speciation in the 'snow-sea ice-seawater' continuum was conducted at a coastal site, near the Australian Casey station (66.40°S; 101.14°E). In the open ocean waters, total Hg (Hg(T)) concentrations varied from 0.63 to 2.76 pmol/L with 'transient-type' vertical profiles and a latitudinal distribution suggesting an atmospheric mercury source south of the Southern Polar Front (SPF) and a surface removal north of the Subantartic Front (SAF). Slightly higher mean Hg(T) concentrations (1.35 ± 0.39 pmol/L) were measured in Antarctic Bottom Water (AABW) compared to Antarctic Intermediate water (AAIW) (1.15 ± 0.22 pmol/L). Labile Hg (Hg(R)) concentrations varied from 0.01 to 2.28 pmol/L, with a distribution showing that the Hg(T) enrichment south of the SPF consisted mainly of Hg(R) (67 ± 23%), whereas, in contrast, the percentage was half that in surface waters north of PFZ (33 ± 23%). Methylated mercury species (MeHg(T)) concentrations ranged from 0.02 to 0.86 pmol/L. All vertical MeHg(T) profiles exhibited roughly the same pattern, with low concentrations observed in the surface layer and increasing concentrations with depth up to an intermediate depth maximum. As for Hg(T), low mean MeHg(T) concentrations were associated with AAIW, and higher ones with AABW. The maximum of MeHg(T) concentration at each station was systematically observed within the oxygen minimum zone, with a statistically significant MeHg(T) vs Apparent Oxygen Utilization (AOU) relationship (p <0.001). The proportion of Hg(T) as methylated species was lower than 5% in the surface waters, around 50% in deep waters below 1000 m, reaching a maximum of 78% south of the SPF. At Casey coastal station Hg(T) and Hg(R) concentrations found in the 'snow-sea ice-seawater' continuum were one order of magnitude higher than those measured in open ocean waters. The distribution of Hg(T) there suggests an atmospheric Hg deposition with snow and a fractionation process during sea ice formation, which excludes Hg from the ice with a parallel Hg enrichment of brine, probably concurring with the Hg enrichment of AABW observed in the open ocean waters. Contrastingly, MeHg(T) concentrations in the sea ice environment were in the same range as in the open ocean waters, remaining below 0.45 pmol/L. The MeHg(T) vertical profile through the continuum suggests different sources, including atmosphere, seawater and methylation in basal ice. Whereas Hg(T) concentrations in the water samples collected between the Antarctic continent and Tasmania are comparable to recent measurements made in the other parts of the World Ocean (e.g., Soerensen et al., 2010; doi:10.1021/es903839n), the Hg species distribution suggests distinct features in the Southern Ocean Hg cycle: (i) a net atmospheric Hg deposition on surface water near the ice edge, (ii) the Hg enrichment in brine during sea ice formation, and (iii) a net methylation of Hg south of the SPF.