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.

Effects of deflazacort versus prednisone on bone mass, body composition, and lipid profile: a randomized, double blind study in kidney transplant patients

To compare the effects of deflazacort (DEFLA) vs. prednisone (PRED) on bone mineral density (BMD), body composition, and lipids, 24 patients with end-stage renal disease were randomized in a double blind design and followed 78 weeks after kidney transplantation. BMD and body composition were assessed using dual energy x-ray absorptiometry. Seventeen patients completed the study. Glucocorticosteroid doses, cyclosporine levels, rejection episodes, and drop-out rates were similar in both groups. Lumbar BMD decreased more in PRED than in DEFLA (P < 0.05), the difference being particularly marked after 24 weeks (9.1 +/- 1.8% vs. 3.0 +/- 2.4%, respectively). Hip BMD decreased from baseline in both groups (P < 0.01), without intergroup differences. Whole body BMD decreased from baseline in PRED (P < 0.001), but not in DEFLA. Lean body mass decreased by approximately 2.5 kg in both groups after 6-12 weeks (P < 0.001), then remained stable. Fat mass increased more (P < 0.01) in PRED than in DEFLA (7.1 +/- 1.8 vs. 3.5 +/- 1.4 kg). Larger increases in total cholesterol (P < 0.03), low density lipoprotein cholesterol (P < 0.01), lipoprotein B2 (P < 0.03), and triglycerides (P = 0.054) were observed in PRED than in DEFLA. In conclusion, using DEFLA instead of PRED in kidney transplant patients is associated with decreased loss of total skeleton and lumbar spine BMD, but does not alter bone loss at the upper femur. DEFLA also helps to prevent fat accumulation and worsening of the lipid profile.

Body composition and fuel metabolism after kidney grafting

Kidney transplant patients display decreased muscle mass and increased fat mass. Whether this altered body composition is due to glucocorticoid induced altered fuel metabolism is unclear. To answer this question, 16 kidney transplant patients were examined immediately after kidney transplantation (12 +/- 4 days, mean +/- SEM) and then during months 2, 5, 11 and 16, respectively, by whole body dual energy X-ray absorptiometry (Hologic QDR 1000W) and indirect calorimetry. Results were compared with those of 16 age, sex and body mass index matched healthy volunteers examined only once. All patients received dietary counselling with a step 1 diet of the American Heart Association and were advised to restrict their caloric intake to the resting energy expenditure plus 30%. Immediately after transplantation, lean mass of the trunk was higher by 7 +/- 1% (P < 0.05) and that of the limbs was lower by more than 10% (P < 0.01) in patients than in controls. In contrast, no difference in fat mass and resting energy expenditure could be detected between patients and controls. During the 16 months of observation, total fat mass increased in male (+4.9 +/- 1.5 kg), but not in female patients (0.1 +/- 0.8 kg). The change in fat mass observed in men was due to an increase in all subregions of the body analysed (trunk, arms+legs as well as head+neck), whereas in women only an increase in head+neck by 9 +/- 2% (P = 0.05) was detected. Body fat distribution remained unchanged in both sexes over the 16 months of observation. Lean mass of the trunk mainly decreased between days 11 and 42 (P < 0.01) and remained stable thereafter. After day 42, lean mass of arms and legs (mostly striated muscle) and head+neck progressively increased over the 14 months of observation by 1.6 +/- 0.6 kg (P < 0.05) and 0.4 +/- 0.1 kg (P < 0.01), respectively. Resting energy expenditure was similar in controls and patients at 42 days (30.0 +/- 0.7 vs. 31.0 +/- 0.9 kcal kg-1 lean mass) and did not change during the following 15 months of observation. However, composition of fuel used to sustain resting energy expenditure in the fasting state was altered in patients when compared with normal subjects, i.e. glucose oxidation was higher by more than 45% in patients (P < 0.01) during the second month after grafting, but gradually declined (P < 0.01) over the following 15 months to values similar to those observed in controls. Protein oxidation was elevated in renal transplant patients on prednisone at first measurement, a difference which tended to decline over the study period. In contrast to glucose and protein oxidation, fat oxidation was lower in patients 42 days after grafting (P < 0.01), but increased by more than 100% reaching values similar to those observed in controls after 16 months of study. Mean daily dose of prednisone per kg body weight correlated with the three components of fuel oxidation (r > 0.93, P < 0.01), i.e. protein, glucose and fat oxidation. These results indicate that in prednisone treated renal transplant patients fuel metabolism is regulated in a dose-dependent manner. Moreover, dietary measures, such as caloric and fat intake restriction as well as increase of protein intake, can prevent muscle wasting as well as part of the usually observed fat accumulation. Furthermore, the concept of preferential upper body fat accumulation as consequence of prednisone therapy in renal transplant patients has to be revised.

ICOMF: Towards a Multi-cloud Ecosystem for Dynamic Resource Composition and Scaling

Modern cloud-based applications and infrastructures may include resources and services (components) from multiple cloud providers, are heterogeneous by nature and require adjustment, composition and integration. The specific application requirements can be met with difficulty by the current static predefined cloud integration architectures and models. In this paper, we propose the Intercloud Operations and Management Framework (ICOMF) as part of the more general Intercloud Architecture Framework (ICAF) that provides a basis for building and operating a dynamically manageable multi-provider cloud ecosystem. The proposed ICOMF enables dynamic resource composition and decomposition, with a main focus on translating business models and objectives to cloud services ensembles. Our model is user-centric and focuses on the specific application execution requirements, by leveraging incubating virtualization techniques. From a cloud provider perspective, the ecosystem provides more insight into how to best customize the offerings of virtualized resources.

Quantitative 1H-magnetic resonance spectroscopy of human brain: Influence of composition and parameterization of the basis set in linear combination model-fitting

Localized short-echo-time (1)H-MR spectra of human brain contain contributions of many low-molecular-weight metabolites and baseline contributions of macromolecules. Two approaches to model such spectra are compared and the data acquisition sequence, optimized for reproducibility, is presented. Modeling relies on prior knowledge constraints and linear combination of metabolite spectra. Investigated was what can be gained by basis parameterization, i.e., description of basis spectra as sums of parametric lineshapes. Effects of basis composition and addition of experimentally measured macromolecular baselines were investigated also. Both fitting methods yielded quantitatively similar values, model deviations, error estimates, and reproducibility in the evaluation of 64 spectra of human gray and white matter from 40 subjects. Major advantages of parameterized basis functions are the possibilities to evaluate fitting parameters separately, to treat subgroup spectra as independent moieties, and to incorporate deviations from straightforward metabolite models. It was found that most of the 22 basis metabolites used may provide meaningful data when comparing patient cohorts. In individual spectra, sums of closely related metabolites are often more meaningful. Inclusion of a macromolecular basis component leads to relatively small, but significantly different tissue content for most metabolites. It provides a means to quantitate baseline contributions that may contain crucial clinical information.

Catch Rates, Composition and Fish Size from Reefs Managed with Periodically-Harvested Closures

Periodically-harvested closures are commonly employed within co-management frameworks to help manage small-scale, multi-species fisheries in the Indo-Pacific. Despite their widespread use, the benefits of periodic harvesting strategies for multi-species fisheries have, to date, been largely untested. We examine catch and effort data from four periodically-harvested reef areas and 55 continuously-fished reefs in Solomon Islands. We test the hypothesis that fishing in periodically-harvested closures would yield: (a) higher catch rates, (b) proportionally more short lived, fast growing, sedentary taxa, and (c) larger finfish and invertebrates, compared to catches from reefs continuously open to fishing. Our study showed that catch rates were significantly higher from periodically-harvested closures for gleaning of invertebrates, but not for line and spear fishing. The family level composition of catches did not vary significantly between open reefs and periodically-harvested closures. Fish captured from periodically-harvested closures were slightly larger, but Trochus niloticus were significantly smaller than those from continuously open reefs. In one case of intense and prolonged harvesting, gleaning catch rates significantly declined, suggesting invertebrate stocks were substantially depleted in the early stages of the open period. Our study suggests periodically-harvested closures can have some short term benefits via increasing harvesting efficiency. However, we did not find evidence that the strategy had substantially benefited multi-species fin-fisheries.

The composition and timing of flower odour emission by wild Petunia axillaris coincide with the antennal perception and nocturnal activity of the pollinator Manduca sexta

In the genus Petunia, distinct pollination syndromes may have evolved in association with bee-visitation (P. integrifolia spp.) or hawk moth-visitation (P. axillaris spp). We investigated the extent of congruence between floral fragrance and olfactory perception of the hawk moth Manduca sexta. Hawk moth pollinated P. axillaris releases high levels of several compounds compared to the bee-pollinated P. integrifolia that releases benzaldehyde almost exclusively. The three dominating compounds in P. axillaris were benzaldehyde, benzyl alcohol and methyl benzoate. In P. axillaris, benzenoids showed a circadian rhythm with an emission peak at night, which was absent from P. integrifolia. These characters were highly conserved among different P. axillaris subspecies and P. axillaris accessions, with some differences in fragrance composition. Electroantennogram (EAG) recordings using flower-blends of different wild Petunia species on female M. sexta antennae showed that P. axillaris odours elicited stronger responses than P. integrifolia odours. EAG responses were highest to the three dominating compounds in the P. axillaris flower odours. Further, EAG responses to odour-samples collected from P. axillaris flowers confirmed that odours collected at night evoked stronger responses from M. sexta than odours collected during the day. These results show that timing of odour emissions by P. axillaris is in tune with nocturnal hawk moth activity and that flower-volatile composition is adapted to the antennal perception of these pollinators.

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.