Universidad Nacional de Cuyo

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.

Ruminant livestock are important sources of human food and global greenhouse gas emissions. Feed degradation and methane formation by ruminants rely on metabolic interactions between rumen microbes and affect ruminant productivity. Rumen and camelid foregut microbial community composition was determined in 742 samples from 32 animal species and 35 countries, to estimate if this was influenced by diet, host species, or geography. Similar bacteria and archaea dominated in nearly all samples, while protozoal communities were more variable. The dominant bacteria are poorly characterised, but the methanogenic archaea are better known and highly conserved across the world. This universality and limited diversity could make it possible to mitigate methane emissions by developing strategies that target the few dominant methanogens. Differences in microbial community compositions were predominantly attributable to diet, with the host being less influential. There were few strong co-occurrence patterns between microbes, suggesting that major metabolic interactions are non-selective rather than specific.

Autophagy is a core molecular pathway for the preservation of cellular and organismal homeostasis. Pharmacological and genetic interventions impairing autophagy responses promote or aggravate disease in a plethora of experimental models. Consistently, mutations in autophagy-related processes cause severe human pathologies. Here, we review and discuss preclinical data linking autophagy dysfunction to the pathogenesis of major human disorders including cancer as well as cardiovascular, neurodegenerative, metabolic, pulmonary, renal, infectious, musculoskeletal, and ocular disorders.

DFTB+ is a versatile community developed open source software package offering fast and efficient methods for carrying out atomistic quantum mechanical simulations. By implementing various methods approximating density functional theory (DFT), such as the density functional based tight binding (DFTB) and the extended tight binding method, it enables simulations of large systems and long timescales with reasonable accuracy while being considerably faster for typical simulations than the respective ab initio methods. Based on the DFTB framework, it additionally offers approximated versions of various DFT extensions including hybrid functionals, time dependent formalism for treating excited systems, electron transport using non-equilibrium Green's functions, and many more. DFTB+ can be used as a user-friendly standalone application in addition to being embedded into other software packages as a library or acting as a calculation-server accessed by socket communication. We give an overview of the recently developed capabilities of the DFTB+ code, demonstrating with a few use case examples, discuss the strengths and weaknesses of the various features, and also discuss on-going developments and possible future perspectives.

Multivesicular bodies (MVBs) are endocytic structures that contain small vesicles formed by the budding of an endosomal membrane into the lumen of the compartment. Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles termed exosomes. K562 cells are a hematopoietic cell line that releases exosomes. The application of monensin (MON) generated large MVBs that were labeled with a fluorescent lipid. Exosome release was markedly enhanced by MON treatment, a Na+/H+ exchanger that induces changes in intracellular calcium (Ca2+). To explore the possibility that the effect of MON on exosome release was caused via an increase in Ca2+, we have used a calcium ionophore and a chelator of intracellular Ca2+. Our results indicate that increasing intracellular Ca2+ stimulates exosome secretion. Furthermore, MON-stimulated exosome release was completely eliminated by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), implying a requirement for Ca2+ in this process. We have observed that the large MVBs generated in the presence of MON accumulated Ca2+ as determined by labeling with Fluo3-AM, suggesting that intralumenal Ca2+ might play a critical role in the secretory process. Interestingly, our results indicate that transferrin (Tf) stimulated exosome release in a Ca2+-dependent manner, suggesting that Tf might be a physiological stimulus for exosome release in K562 cells.

and to screen large chemical libraries for putative ICD inducers, based on a high-content, high-throughput platform that we recently developed. Such a platform allows for the detection of multiple DAMPs, like cell surface-exposed calreticulin, extracellular ATP and high mobility group box 1 (HMGB1), and/or the processes that underlie their emission, such as endoplasmic reticulum stress, autophagy and necrotic plasma membrane permeabilization. We surmise that this technology will facilitate the development of next-generation anticancer regimens, which kill malignant cells and simultaneously convert them into a cancer-specific therapeutic vaccine.

Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic components and organelles in a vacuole called an autophagosome that finally fuses with the lysosome. Rab7 is a member of the Rab family involved in transport to late endosomes and in the biogenesis of the perinuclear lysosome compartment. To assess the role of Rab7 in autophagy we stably transfected CHO cells with wild-type pEGFP-Rab7, and the mutants T22N (GDP form) and Q67L (GTP form). Autophagy was induced by amino acid starvation and the autophagic vacuoles were labeled with monodansylcadaverine. By fluorescence microscopy we observed that Rab7wt and the active mutant Rab7Q67L were associated with ring-shaped vesicles labeled with monodansylcadaverine indicating that these Rab proteins associate with the membrane of autophagic vesicles. As expected, in cells transfected with the negative mutant Rab7T22N the protein was diffusely distributed in the cytosol. However, upon induction of autophagy by amino acid starvation or by rapamycin treatment this mutant clearly decorated the monodansylcadaverine-labeled vesicles. Furthermore, a marked increase in the size of the monodansylcadaverine-labeled vacuoles induced by starvation was observed by overexpression of the inactive mutant T22N. Similarly, there was an increase in the size of vesicles labeled with LC3, a protein that specifically localizes on the autophagosomal membrane. Taken together the results indicate that a functional Rab7 is important for the normal progression of autophagy.

BACKGROUND: Ecologists and evolutionary biologists are becoming increasingly interested in networks as a framework to study plant-animal mutualisms within their ecological context. Although such focus on networks has brought about important insights into the structure of these interactions, relatively little is still known about the mechanisms behind these patterns. SCOPE: The aim in this paper is to offer an overview of the mechanisms influencing the structure of plant-animal mutualistic networks. A brief summary is presented of the salient network patterns, the potential mechanisms are discussed and the studies that have evaluated them are reviewed. This review shows that researchers of plant-animal mutualisms have made substantial progress in the understanding of the processes behind the patterns observed in mutualistic networks. At the same time, we are still far from a thorough, integrative mechanistic understanding. We close with specific suggestions for directions of future research, which include developing methods to evaluate the relative importance of mechanisms influencing network patterns and focusing research efforts on selected representative study systems throughout the world.

Philipp Simon, Massimo Iorizzo, Allen Van Deynze and colleagues report the high-quality assembly of the carrot genome, providing an important resource for crop improvement. They find a candidate gene that regulates carotenoid accumulation and gain further insights into asterid genome evolution, including characterization of two new polyploidization events. We report a high-quality chromosome-scale assembly and analysis of the carrot (Daucus carota) genome, the first sequenced genome to include a comparative evolutionary analysis among members of the euasterid II clade. We characterized two new polyploidization events, both occurring after the divergence of carrot from members of the Asterales order, clarifying the evolutionary scenario before and after radiation of the two main asterid clades. Large- and small-scale lineage-specific duplications have contributed to the expansion of gene families, including those with roles in flowering time, defense response, flavor, and pigment accumulation. We identified a candidate gene, DCAR_032551, that conditions carotenoid accumulation (Y) in carrot taproot and is coexpressed with several isoprenoid biosynthetic genes. The primary mechanism regulating carotenoid accumulation in carrot taproot is not at the biosynthetic level. We hypothesize that DCAR_032551 regulates upstream photosystem development and functional processes, including photomorphogenesis and root de-etiolation.

Patellar instability is defined by clinical and radiologic criteria. The surgical treatment is aimed at restoring the congruence of the patellofemoral articulation and correcting extensor mechanism malalignment, to prevent recurrence of dislocation. The standard soft-tissue procedures are lateral release and vastus medialis advancement and medial patello femoral ligament plasty. Bony procedures are frequently performed in addition to soft-tissue surgery, to realign the extensor mechanism by means of tibial tubercle medialization, or to correct the patellar index in patella alta. In a smaller number of patients, the trochlea may be reshaped, by elevating the lateral trochlear facet or by lowering the floor of the sulcus. The morphologic abnormalities encountered are studied and quantified in the light of preoperative investigations (conventional radiographs and computed tomography), and addressed at surgery using the technique or techniques most appropriate for the management of the individual patient's pattern.

Reproducible climate reconstructions of the Common Era (1 CE to present) are key to placing industrial-era warming into the context of natural climatic variability. Here we present a community-sourced database of temperature-sensitive proxy records from the PAGES2k initiative. The database gathers 692 records from 648 locations, including all continental regions and major ocean basins. The records are from trees, ice, sediment, corals, speleothems, documentary evidence, and other archives. They range in length from 50 to 2000 years, with a median of 547 years, while temporal resolution ranges from biweekly to centennial. Nearly half of the proxy time series are significantly correlated with HadCRUT4.2 surface temperature over the period 1850-2014. Global temperature composites show a remarkable degree of coherence between high- and low-resolution archives, with broadly similar patterns across archive types, terrestrial versus marine locations, and screening criteria. The database is suited to investigations of global and regional temperature variability over the Common Era, and is shared in the Linked Paleo Data (LiPD) format, including serializations in Matlab, R and Python.

Autophagy is a normal degradative pathway that involves the sequestration of cytoplasmic portions and intracellular organelles in a membrane vacuole called the autophagosome. These vesicles fuse with lysosomes and the sequestered material is degraded. Owing to the complexity of the autophagic pathway and to its inaccessibility to external probes, little is known about the molecular mechanisms that regulate autophagy in higher eukaryotic cells. We used the autofluorescent drug monodansylcadaverine (MDC), a specific autophagolysosome marker to analyze at the molecular level the machinery involved in the autophagic process. We have developed a morphological and biochemical assay to study authophagy in living cells based on the incorporation of MDC. With this assay we observed that the accumulation of MDC was specifically induced by amino acid deprivation and was inhibited by 3-methlyadenine, a classical inhibitor of the autophagic pathway. Additionally, wortmannin, an inhibitor of PI3-kinases that blocks autophagy at an early stage, inhibited the accumulation of MDC in autophagic vacuoles. We also found that treatment of the cells with N-ethylmaleimide (NEM), an agent known to inhibit several vesicular transport events, completely blocked the incorporation of MDC, suggesting that an NEM-sensitive protein is required for the formation of autophagic vacuoles. Conversely, vinblastine, a microtubule depolymerizing agent that induces the accumulation of autophagic vacuoles by preventing their degradation, increased the accumulation of MDC and altered the distribution and size of the autophagic vacuoles. Our results indicate that in the presence of vinblastine very large MDC-vacuoles accumulated mainly under starvation conditions, indicating that the expansion of autophagosomes is upregulated by amino acid deprivation. Furthermore, these MDC-vacuoles were labeled with LC3, one of the mammalian homologues of the yeast protein Apg8/Aut7 that plays an important role in autophagosome formation.

Multivesicular bodies (MVBs) are membranous structures within 60-100 nm diameter vesicles accumulate. MVBs are generated after invagination and pinching off of the endosomal membrane in the lumen of the vacuole. In certain cell types, fusion of MVBs with the plasma membrane results in the release of the internal vesicles called exosomes. In this report we have examined how an increase in cytosolic calcium affects the development of MVBs and exosome release in K562 cells overexpressing GFP-Rab11 wt or its mutants. In cells overexpressing the Rab11Q70 L mutant or Rab11 wt, an increase in the cytosolic calcium concentration induced by monensin caused a marked enlargement of the MVBs. This effect was abrogated by the membrane permeant calcium chelator BAPTA-AM. We also examined the behavior of MVBs in living cells by time lapse confocal microscopy. Many MVBs, decorated by wt or Q70L mutant GFP-Rab11, were docked and ready to fuse in the presence of a calcium chelator. This observation suggests that Rab11 is acting in the tethering/docking of MVBs to promote homotypic fusion, but that the final fusion reaction requires the presence of calcium. Additionally, a rise in intracellular calcium concentration enhanced exosome secretion in Rab11 wt overexpressing cells and reversed the inhibition of the mutants. The results suggest that both Rab11 and calcium are involved in the homotypic fusion of MVBs.

During maturation, reticulocytes lose some membrane proteins that are not required on the mature red cell surface. The proteins are released into the extracellular medium associated with vesicles that are formed by budding of the endosomal membrane into the lumen of the compartment; this process results in the formation of multivesicular bodies (MVBs). Fusion of MVBs with the plasma membrane results in secretion of the small internal vesicles, termed exosomes. K562 cells release exosomes with similar characteristics to reticulocyte exosomes, in particular the transferrin receptor (TfR) is found associated with the vesicles. Interestingly, this cell line has been shown to possess high amounts of Rab11 compared with other Rab proteins. To assess the regulation of transferrin receptor release via exosome secretion by Rab11 in this cell type, K562 cells were stably transfected with GFP-Rab11wt or the GTP- and GDP-locked mutants. The distribution of the proteins was assessed by fluorescence microscopy. Transferrin recycling and the number of TfRs present on the surface of the transfected cells were reduced by overexpression of either Rab11wt or the mutants. The amount of released exosomes was analyzed by measuring different molecular markers present on these vesicles either biochemically or by western blot. Overexpression of the dominant-negative mutant Rab11S25N inhibited exosome release, whereas the secretion of exosomes was slightly stimulated in cells transfected with Rab11wt. Taken together, the results demonstrate that in K562 cells Rab11 modulates the exosome pathway although the exact step involved is still not known.

Morphological and biochemical studies have shown that autophagosomes fuse with endosomes forming the so-called amphisomes, a prelysosomal hybrid organelle. In the present report, we have analyzed this process in K562 cells, an erythroleukemic cell line that generates multivesicular bodies (MVBs) and releases the internal vesicles known as exosomes into the extracellular medium. We have previously shown that in K562 cells, Rab11 decorates MVBs. Therefore, to study at the molecular level the interaction of MVBs with the autophagic pathway, we have examined by confocal microscopy the fate of MVBs in cells overexpressing green fluorescent protein (GFP)-Rab11 and the autophagosomal protein red fluorescent protein-light chain 3 (LC3). Autophagy inducers such as starvation or rapamycin caused an enlargement of the vacuoles decorated with GFP-Rab11 and a remarkable colocalization with LC3. This convergence was abrogated by a Rab11 dominant negative mutant, indicating that a functional Rab11 is involved in the interaction between MVBs and the autophagic pathway. Interestingly, we presented evidence that autophagy induction caused calcium accumulation in autophagic compartments. Furthermore, the convergence between the endosomal and the autophagic pathways was attenuated by the Ca2+ chelator acetoxymethyl ester (AM) of the calcium chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), indicating that fusion of MVBs with the autophagosome compartment is a calcium-dependent event. In addition, autophagy induction or overexpression of LC3 inhibited exosome release, suggesting that under conditions that stimulates autophagy, MVBs are directed to the autophagic pathway with consequent inhibition in exosome release.

The structure of mutualistic networks is likely to result from the simultaneous influence of neutrality and the constraints imposed by complementarity in species phenotypes, phenologies, spatial distributions, phylogenetic relationships, and sampling artifacts. We develop a conceptual and methodological framework to evaluate the relative contributions of these potential determinants. Applying this approach to the analysis of a plant-pollinator network, we show that information on relative abundance and phenology suffices to predict several aggregate network properties (connectance, nestedness, interaction evenness, and interaction asymmetry). However, such information falls short of predicting the detailed network structure (the frequency of pairwise interactions), leaving a large amount of variation unexplained. Taken together, our results suggest that both relative species abundance and complementarity in spatiotemporal distribution contribute substantially to generate observed network patters, but that this information is by no means sufficient to predict the occurrence and frequency of pairwise interactions. Future studies could use our methodological framework to evaluate the generality of our findings in a representative sample of study systems with contrasting ecological conditions.

Grape pomaces (GPs) are characterized by high contents of phenolics due to an incomplete extraction during the winemaking process. These phenolics are secondary plant metabolites with potential beneficial effects on human health because of their antioxidant activity and antimicrobial, antiviral, and anti-inflammatory properties. Therefore, GP constitutes an inexpensive source for the extraction of phytochemicals that can be used in the pharmaceutical, cosmetic, and food industries. As a result of the increased attention to sustainability of agricultural practices, efforts have been made to use GP in different fields of industry. Thus, it is necessary to have efficient extraction techniques to achieve good recoveries of compounds. In this respect, sensitive and selective analytical methods have been tried for the characterization of phenolic extracts. This review summarizes the most recent developments in the extraction of polyphenols from GPs. Furthermore, the techniques used for characterization of extracts are explained, with emphasis on sample preparation, separation, and analysis of phenolics. Finally, the possible applications of GP extracts in diverse biotechnological fields are also discussed.

Large earthquakes produce crustal deformation that can be quantified by geodetic measurements, allowing for the determination of the slip distribution on the fault. We used data from Global Positioning System (GPS) networks in Central Chile to infer the static deformation and the kinematics of the 2010 moment magnitude (M(w)) 8.8 Maule megathrust earthquake. From elastic modeling, we found a total rupture length of ~500 kilometers where slip (up to 15 meters) concentrated on two main asperities situated on both sides of the epicenter. We found that rupture reached shallow depths, probably extending up to the trench. Resolvable afterslip occurred in regions of low coseismic slip. The low-frequency hypocenter is relocated 40 kilometers southwest of initial estimates. Rupture propagated bilaterally at about 3.1 kilometers per second, with possible but not fully resolved velocity variations.

The joining of different genomes in allotetraploids played a major role in plant evolution, but the molecular implications of this event are poorly understood. In synthetic allotetraploids of Arabidopsis and Cardaminopsis arenosa, we previously demonstrated the occurrence of frequent gene silencing. To explore the involvement of epigenetic phenomena, we investigated the occurrence and effects of DNA methylation changes. Changes in DNA methylation patterns were more frequent in synthetic allotetraploids than in the parents. Treatment with 5-aza-2'-deoxycytidine, an inhibitor of DNA methyltransferase, resulted in the development of altered morphologies in the synthetic allotetraploids, but not in the parents. We profiled mRNAs in control and 5-aza-2'-deoxycytidine-treated parents and allotetraploids by amplified fragment length polymorphism-cDNA. We show that DNA demethylation induced and repressed two different transcriptomes. Our results are consistent with the hypothesis that synthetic allotetraploids have compromised mechanisms of epigenetic gene regulation.

Production of phytohormones is one of the main mechanisms to explain the beneficial effects of plant growth-promoting rhizobacteria (PGPR) such as Azospirillum sp. The PGPRs induce plant growth and development, and reduce stress susceptibility. However, little is known regarding the stress-related phytohormone abscisic acid (ABA) produced by bacteria. We investigated the effects of Azospirillum brasilense Sp 245 strain on Arabidopsis thaliana Col-0 and aba2-1 mutant plants, evaluating the morphophysiological and biochemical responses when watered and in drought. We used an in vitro-grown system to study changes in the root volume and architecture after inoculation with Azospirillum in Arabidopsis wild-type Col-0 and on the mutant aba2-1, during early growth. To examine Arabidopsis development and reproductive success as affected by the bacteria, ABA and drought, a pot experiment using Arabidopsis Col-0 plants was also carried out. Azospirillum brasilense augmented plant biomass, altered root architecture by increasing lateral roots number, stimulated photosynthetic and photoprotective pigments and retarded water loss in correlation with incremented ABA levels. As well, inoculation improved plants seed yield, plants survival, proline levels and relative leaf water content; it also decreased stomatal conductance, malondialdehyde and relative soil water content in plants submitted to drought. Arabidopsis inoculation with A. brasilense improved plants performance, especially in drought.