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.     

The marker SCK13<Subscript>603</Subscript> associated with resistance to ascochyta blight in chickpea is located in a region of a putative retrotransposon

The sequence characterized amplified region (SCAR) marker SCK13(603), associated with ascochyta blight resistance in a chickpea recombinant inbred line (RIL) population, was used as anchored sequence for genome walking. The PCRs performed in the walking steps to walk in the same direction produced eight bands in 5' direction and five bands in 3' direction with a length ranking from 530 to 2,871 bp. The assembly of the bands sequences along with the sequence of SCK13(603) resulted in 7,815 bp contig. Blastn analyses showed stretches of DNA sequence mainly distributed from the nucleotides 1,500 to 4,500 significantly similar to Medicago truncatula genomic DNA. Three open reading frames (ORFs) were identified and blastp analysis of predicted amino acids sequences revealed that ORF1, ORF2 and ORF3 had significant similarity to a CCHC zinc finger protein, to an integrase, and to a precursor of the glucoamylase s1/s2, respectively, from M. truncatula. The high homology of the putative proteins derived from ORF1 and ORF2 with retrotransposon proteins and the prediction of the existence of conserved domains usually present in retrotransposon proteins indicate that the marker SCK13(603) is located in a region of a putative retrotransposon. The information generated in this study has contributed to increase the knowledge of this important region for blight resistance in chickpea.     

Synthesis, cytotoxic evaluation, and DNA binding of novel thiazolo[5,4-b]quinoline derivatives

A series of novel alkylamino and 9-anilinothiazolo[5,4-b]quinolines were synthesized as potential antitumoral agents. The in vitro cytotoxicity of these compounds was evaluated on several cell lines. The inclusion of electron-withdrawn/acceptor hydrogen-bond groups at position 3' of the anilino ring and the presence of an alkylamino chain on the tricyclic framework (regardless of its position) seem to be structural features relevant to cytotoxic activity.     

A model for small‐scale seed bank and standing vegetation connection along time

Since inter‐annual climatic variability influences composition and structure of seed bank and extant vegetation, it is expected that it also affects the relationship between both compartments at small scales along time. We hypothesize that seed bank and aboveground vegetation are closely and sequentially linked at small spatial and time scales. We conducted a field spatially explicit investigation to explore the spatial and temporal relationship between both compartments. Abundance, composition and spatial structure at different small‐scales of seed bank and aboveground vegetation were analysed through 100 permanent plots during two consecutive (dry and wet) periods of growth. Following a conceptual transition path model, we analysed changes in correlation values in composition and spatial aggregation between both compartments along time including seasonal variations of seed bank. Shape of spatial structures were evaluated using partial Mantel correlograms. Annuals and perennials guilds were studied separately. During the wet year, annuals increased their aerial spatial aggregation and cover, whereas the opposite happened for perennials. Density and spatial aggregation of the seed bank increased for both guilds in the two seasons following the rain period, especially in annuals. The clumped structure of the transient seed bank is a consequence of the addition of spatial structure of extant vegetation and the persistent seed bank. Expression of the persistent seed bank is much lower during the dry period for the annual guild. In spite of the strong yearly variability, the community maintained a highly structured spatiotemporal pattern. The mechanisms promoting this stability differed for annual and perennial guilds. Temporal persistence in perennials relied mainly in established plants longevity, whereas annual guild persistence depended solely on seed bank. This tight structure was coherent with the existence of successional dynamics in the community, although persistent seed bank could moderate the pace. Longer term studies of seed bank–standing vegetation dynamics are required to fully understand this process.     

Actin bundling via LIM domains

The LIM domain is defined as a protein-protein interaction module involved in the regulation of diverse cellular processes including gene expression and cytoskeleton organization. We have recently shown that the tobacco WLIM1, a two LIM domain-containing protein, is able to bind to, stabilize and bundle actin filaments, suggesting that it participates to the regulation of actin cytoskeleton structure and dynamics. In the December issue of the Journal of Biological Chemistry we report a domain analysis that specifically ascribes the actin-related activities of WLIM1 to its two LIM domains. Results suggest that LIM domains function synergistically in the full-length protein to achieve optimal activities. Here we briefly summarize relevant data regarding the actin-related properties/functions of two LIM domain-containing proteins in plants and animals. In addition, we provide further evidence of cooperative effects between LIM domains by transiently expressing a chimeric multicopy WLIM1 protein in BY2 cells.Key words: Actin-binding proteins, actin-bundling, cysteine-rich proteins, cytoskeleton, LIM domainThe LIM domain is a ≈55 amino acid peptide domain that was first identified in 1990 as a common cystein-rich sequence found in the three homeodomain proteins LIN-11, Isl1 and MEC-3. It has since been found in a wide variety of eukaryotic proteins of diverse functions. Animals possess several families of LIM proteins, with members containing 1–5 LIM domains occasionally linked to other catalytic or protein-binding domains such as homeodomain, kinase and SH3 domains. In contrast, plants only possess two distinct sets of LIM proteins. One is plant-specific and has not been functionally characterized yet. The other one comprises proteins that exhibit the same overall structure as the animal cystein rich proteins (CRPs), i.e., two very similar LIM domains separated by a ≈50 amino acid-long interLIM domain and a relatively short and variable C-terminal domain (Fig. 1A). The mouse CRP2 protein was the first CRP reported to interact directly with actin filaments (AF) and to stabilize the latter.¹ Identical observations were subsequently described for the chicken CRP1 and tobacco WLIM1 proteins.^2,3 In addition, these two proteins were shown to arrange AF into cables both in vitro and in vivo and thus join the list of actin bundlers.Open in a separate windowFigure 1Domain maps for wild-type WLIM1 (A) and GFP-fused chimeric 3xWLIM1 (B). A. WLIM1 basically comprises a short N-terminal domain (Nt), two LIM domains (LIM1 and LIM2), an interLIM spacer (IL) and a C-terminal domain (Ct). B. 3xWLIM1 consists of three tandem WLIM1 copies. This chimeric protein has been fused in C-terminus to GFP and transiently expressed in tobacco BY2 cells.To identify the peptide domains of WLIM1 responsible for its actin-related properties/activities, we generated domain-deleted and single domain variants and submitted them to a series of in vivo and in vitro assays.⁴ Localization experiments established that both LIM domains are required to efficiently target the actin cytoskeleton in tobacco BY2 cells. High-speed (200,000 g) cosedimentation data confirmed that the actin-binding activity of WLIM1 relies on its LIM domains. Indeed, the deletion of either the first or the second LIM domain respectively resulted in a 5-fold and 10-fold decrease of the protein affinity for AF. Importantly, each single LIM domain was found able to interact with AF in an autonomous manner, although with a reduced affinity compared to the wild-type WLIM1. Low-speed (12,500 g) cosedimentation data and electron microscopy observations revealed that the actin bundling activity of WLIM1 is also triggered by its LIM domains. Surprisingly each single LIM domain was able to bundle AF in an autonomous manner, suggesting that WLIM1 has two discrete actin-bundling sites. However, the bundles induced by the variants containing only one LIM domain, i.e., LIM domain-deleted mutants and single LIM domains, differed from those induced by the full-length WLIM1. They appeared more wavy and loosely packed and formed only at relatively high protein:actin ratios. Together these data suggest that LIM domains are autonomous actin-binding and -bundling modules that function in synergy in wild-type WLIM1 to achieve optimal activities.To further assess the mechanism of cooperation between the LIM domains of plant CRP-related proteins, we generated a chimeric protein composed of three WLIM1 copies in tandem (3 × WLIM1, Fig. 1B), and transiently expressed it as a GFP-fusion in tobacco BY2 cells. We anticipated that such a six LIM domain-containing protein displays an even higher actin-bundling activity. (Fig. 2A) shows the typical actin cytoskeleton pattern in an expanding BY2 cell as visualized using the actin marker GFP-fABD2.⁵ As previously reported by Sheahan et al.,⁵ GFP-fABD2 decorated dense, transversely oriented, cortical networks as well as transvacuolar strands connecting the subcortical-perinuclear region to the cortex. Ectopic expression of WLIM1-GFP (BY2 cells normally do not express the WLIM1 gene) induced moderate but perceptible modifications of the actin cytoskeleton structure (Fig. 2B). Most AF are arranged in bundles thicker than those observed in GFP-fABD2 expressing cells and fine AF arrays are less frequently observed. As expected, this phenotype was significantly enhanced in cells transformed with the 3xWLIM1-GFP protein (Fig. 2C). Indeed, cells were almost devoided of fine AF arrays and exhibited very thick actin cables (Fig. 2C) that, at times (≈30 %), form atypical long looped structures (Fig. 2D). The appearance of such structures may result from the increase of cable stability and thickness induced by the 3xWLIM1-GFP protein, as these parameters are likely to determine, at least partially, the maximal length of actin bundles. Together the present observations support earlier data showing that LIM domains work in concert in LIM proteins to regulate actin bundling in plant cells. Strikingly, vertebrate and plant CRPs invariably contain two LIM domains. The lack, in these organisms, of CRP-related proteins combining more than two LIM domains may be explained by the fact that very thick cables, such as those induced by the artificial 3xWLIM1, may be too stable structures incompatible with the necessary high degree of actin cytoskeleton plasticity. As an exception, a muscle CRP-related protein with five LIM domains (Mlp84B) has been identified in Drosophila.⁶ However, rather than decorating actin filaments in an homogenous manner, this protein has been found to concentrate in a specialized region of the Z-discs where it stabilizes, in concert with D-titin, muscle sarcomeres.⁷Open in a separate windowFigure 2Typical actin cytoskeleton patterns in tobacco BY2 cells that have been transiently transformed, using a particle gun, with GFP-fABD2 (A), WLIM1-GFP (B), and 3xWLIM1-GFP (C and D). For each construct, more than 60 cells were analyzed by confocal microscopy. In the case of 3xWLIM1-GFP, two prevalent patterns have been observed (C and D). Bars = 20 µm.The relatively well conserved spacer length (≈50 amino acids) that separates the two LIM domains in vertebrate CRPs and related plant LIM proteins remains an intriguing feature the importance of which in actin cable organization remains to be established. Using electron microscopy we are currently evaluating the effects of the modification of the interLIM domain length on the structural properties of actin cables.     

Synthesis,crystal structure and magnetic properties of the spin crossover system [Fe(pq)3]2+

Three new compounds formulated (ClO₄)₂[Fe(pq)₃] (1), (BF₄)₂[Fe(pq)₃] · EtOH (2) and {(ClO₄)[MnCr(C₂O₄)₃][Fe(pq)₂(H₂O)₂]} (3), where pq is 2,2′-pyridylquinoline, have been synthesised and characterised. Despite the different crystal packing exhibited by 1 and 2, the cationic species [Fe(pq)₃]²⁺ are structurally quite similar. At 293 K, the Fe–N bond lengths are characteristic of the iron(II) in the high-spin state. In contrast to 1, 2 undergoes a continuous spin transition. Indeed, at 95 K its structure experiences a noticeable change in the Fe–N bonds and angles, i.e. the Fe–N bonds shorten by 0.194 Å on the average. The magnetic behaviour confirms that 1 is fully high-spin in the 4–300 K temperature range while 2 shows a spin transition centred at T_1/2 = 150 K. The corresponding enthalpy, entropy and interaction parameter are ΔH = 7.49 kJ mol^?1, ΔS = 50 J K^?1 mol^?1and Γ = 1.35 kJ mol^?1. Compound 3 has been obtained as a microcrystalline powder. The magnetic properties of 3 point at the occurrence of ferromagnetic coupling below 100 K and the onset of a ferromagnetic ordering below 10 K (Weiss constant equal to 6.8 K). The Mössbauer spectra of 3 show the occurrence of a magnetic order at T ? 4.2 K.     

Detection of Tissue Factor Antigen and Coagulation Activity in Coronary Artery Thrombi Isolated from Patients with ST-Segment Elevation Acute Myocardial Infarction

Introduction

Although ruptured atherosclerotic plaques have been extensively analyzed, the composition of thrombi causing arterial occlusion in patients with ST-segment elevation acute myocardial infarction has been less thoroughly investigated. We sought to investigate whether coagulant active tissue factor can be retrieved in thrombi of patients with STEMI undergoing primary percutaneous coronary intervention.

Methods

Nineteen patients with ST-segment elevation acute myocardial infarction referred for primary percutaneous coronary intervention were enrolled in this study. Coronary thrombi aspirated from coronary arteries were routinely processed for paraffin embedding and histological evaluation (4 patients) or immediately snap frozen for evaluation of tissue factor activity using a modified aPTT test (15 patients). Immunoprecipitation followed by immunoblotting was also performed in 12 patients.

Results

Thrombi aspirated from coronary arteries showed large and irregular areas of tissue factor staining within platelet aggregates, and in close contact with inflammatory cells. Some platelet aggregates stained positive for tissue factor, whereas others did not. Monocytes consistently stained strongly for tissue factor, neutrophils had a more variable and irregular tissue factor staining, and red blood cells did not demonstrate staining for tissue factor. Median clotting time of plasma samples containing homogenized thrombi incubated with a monoclonal antibody that specifically inhibits tissue factor-mediated coagulation activity (mAb 5G9) were significantly longer than their respective controls (88.9 seconds versus 76.5 seconds, respectively; p<0.001). Tissue factor was also identified by immunoprecipitation in 10 patients, with significant variability among band intensities.

Conclusions

Active tissue factor is present in coronary artery thrombi of patients with ST-segment elevation acute myocardial infarction, suggesting that it contributes to activate the coagulation cascade ensuing in coronary thrombosis.     

New Arabidopsis thaliana Cytochrome c Partners: A Look Into the Elusive Role of Cytochrome c in Programmed Cell Death in Plants

Programmed cell death is an event displayed by many different organisms along the evolutionary scale. In plants, programmed cell death is necessary for development and the hypersensitive response to stress or pathogenic infection. A common feature in programmed cell death across organisms is the translocation of cytochrome c from mitochondria to the cytosol. To better understand the role of cytochrome c in the onset of programmed cell death in plants, a proteomic approach was developed based on affinity chromatography and using Arabidopsis thaliana cytochrome c as bait. Using this approach, ten putative new cytochrome c partners were identified. Of these putative partners and as indicated by bimolecular fluorescence complementation, nine of them bind the heme protein in plant protoplasts and human cells as a heterologous system. The in vitro interaction between cytochrome c and such soluble cytochrome c-targets was further corroborated using surface plasmon resonance. Taken together, the results obtained in the study indicate that Arabidopsis thaliana cytochrome c interacts with several distinct proteins involved in protein folding, translational regulation, cell death, oxidative stress, DNA damage, energetic metabolism, and mRNA metabolism. Interestingly, some of these novel Arabidopsis thaliana cytochrome c-targets are closely related to those for Homo sapiens cytochrome c (Martínez-Fábregas et al., unpublished). These results indicate that the evolutionarily well-conserved cytosolic cytochrome c, appearing in organisms from plants to mammals, interacts with a wide range of targets on programmed cell death. The data have been deposited to the ProteomeXchange with identifier PXD000280.Programmed cell death (PCD)¹ is a fundamental event for the development of multicellular organisms and the homeostasis of their tissues. It is an evolutionarily conserved mechanism present in organisms ranging from yeast to mammals (1–3).In mammals, cytochrome c (Cc) and dATP bind to apoptosis protease-activating factor-1 (Apaf-1) in the cytoplasm, a process leading to the formation of the Apaf-1/caspase-9 complex known as apoptosome. This apoptosome subsequently activates caspases-3 and -7 (4, 5). In other organisms, such as Caenorhabditis elegans or Drosophila melanogaster, however, Cc is not essential for the assembly and activation of the apoptosome (6) despite the presence of proteins homologous to Apaf-1—cell death abnormality-4 (CED-4) in C. elegans and Drosophila Apaf-1-related killer (Dark) in D. melanogaster—which have been found to be essential for caspase cascade activation. Furthermore, other organisms such as Arabidopsis thaliana lack Apaf-1 (7). In fact, only highly distant caspase homologues (metacaspases) (8, 9), serine proteases (saspases) (10), phytaspases (11) and VEIDases (12–14) with caspase-like activity have been detected in plants; however, their targets remain veiled and whether they are activated by Cc remains unclear.Intriguingly, the release of Cc from mitochondria into the cytoplasm during the onset of PCD is an evolutionarily conserved event found in organisms ranging from yeast (15) and plants (16) to flies (17), and mammals (18). However, understanding of the roles of this phenomenon in different species can be said to be uneven at best. In fact, the release of Cc from mitochondria has thus far been considered a random event in all organisms, save mammals. Thus, the participation of Cc in the onset and progression of PCD needs to be further elucidated.Even in the case of mammals, the role(s) of Cc in the cytoplasm during PCD remain(s) controversial. Recently, new putative functions of Cc, going beyond the already-established apoptosome assembly process, have been proposed in the nucleus (19, 20) and the endoplasmic reticulum (21–23). Neither these newly proposed functions nor other arising functions, such as oxidative stress (24), are as yet fully understood. This current state of affairs demands deeper exploration of the additional roles played by Cc in nonmammalian species.In this study, putative novel Cc-partners involved in plant PCD were identified. For this identification, a proteomic approach was employed based on affinity chromatography and using Cc as bait. The Cc-interacting proteins were identified using nano-liquid chromatography tandem mass spectrometry (NanoLC-MS/MS). These Cc-partners were then further confirmed in vivo through bimolecular fluorescence complementation (BiFC) in A. thaliana protoplasts and human HEK293T cells, as a heterologous system. Finally, the Cc-GLY2, Cc-NRP1 and Cc-TCL interactions were corroborated in vitro using surface plasmon resonance (SPR).These results indicate that Cc is able to interact with targets in the plant cell cytoplasm during PCD. Moreover, they provide new ways of understanding why Cc release is an evolutionarily well-conserved event, and allow us to propose Cc as a signaling messenger, which somehow controls different essential events during PCD.     

Chagas disease: control,elimination and eradication. Is it possible?

From an epidemiological point of view, Chagas disease and its reservoirs and vectors can present the following characteristics: (i) enzooty, maintained by wild animals and vectors, with broad occurrence from southern United States of America (USA) to southern Argentina and Chile (42ºN 49ºS), (ii) anthropozoonosis, when man invades the wild ecotope and becomes infected with Trypanosoma cruzi from wild animals or vectors or when the vectors and wild animals, especially marsupials, invade the human domicile and infect man, (iii) zoonosis-amphixenosis and exchanged infection between animals and humans by domestic vectors in endemic areas and (iv) zooanthroponosis, infection that is transmitted from man to animals, by means of domestic vectors, which is the rarest situation in areas endemic for Chagas disease. The characteristics of Chagas disease as an enzooty of wild animals and as an anthropozoonosis are seen most frequently in the Brazilian Amazon and in the Pan-Amazon region as a whole, where there are 33 species of six genera of wild animals: Marsupialia, Chiroptera, Rodentia, Edentata (Xenarthra), Carnivora and Primata and 27 species of triatomines, most of which infected with T. cruzi . These conditions place the resident populations of this area or its visitors - tourists, hunters, fishermen and especially the people whose livelihood involves plant extraction - at risk of being affected by Chagas disease. On the other hand, there has been an exponential increase in the acute cases of Chagas disease in that region through oral transmission of T. cruzi , causing outbreaks of the disease. In four seroepidemiological surveys that were carried out in areas of the microregion of the Negro River, state of Amazonas, in 1991, 1993, 1997 and 2010, we found large numbers of people who were serologically positive for T. cruzi infection. The majority of them and/or their relatives worked in piassava extraction and had come into contact with and were stung by wild triatomines in that area. Finally, a characteristic that is greatly in evidence currently is the migration of people with Chagas disease from endemic areas of Latin America to non-endemic countries. This has created a new dilemma for these countries: the risk of transmission through blood transfusion and the onus of controlling donors and treating migrants with the disease. As an enzooty of wild animals and vectors, and as an anthropozoonosis, Chagas disease cannot be eradicated, but it must be controlled by transmission elimination to man.     

Involvement of Autonomic Nervous Activity Changes in Gastroesophageal Reflux in Neonates during Sleep and Wakefulness

Background

It has been suggested that disturbed activity of the autonomic nervous system is one of the factors involved in gastroesophageal reflux (GER) in adults. We sought to establish whether transient ANS dysfunction (as assessed by heart rate variability) is associated with the occurrence of GER events in neonates during sleep and wakefulness.

Methods

Nineteen neonates with suspected GER underwent simultaneous, synchronized 12-hour polysomnography and esophageal multichannel impedance-pH monitoring. We compared changes in HRV parameters during three types of periods (control and prior to and during reflux) with respect to the vigilance state.

Results

The vigilance state influenced the distribution of GER events (P<0.001), with 53.4% observed during wakefulness, 37.6% observed during active sleep and only 9% observed during quiet sleep. A significant increase in the sympathovagal ratio (+32%, P=0.013) was observed in the period immediately prior to reflux (due to a 15% reduction in parasympathetic activity (P=0.017)), relative to the control period. This phenomenon was observed during both wakefulness and active sleep.

Conclusion

Our results showed that GER events were preceded by a vigilance-state-independent decrease in parasympathetic tone. This suggests that a pre-reflux change in ANS activity is one of the factors contributing to the mechanism of reflux in neonates.