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. 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. Multivesicular bodies (MVBs) 1The abbreviations used are: MVBs, multivesicular bodies; MON, monensin; Tf, transferrin; TfR, Tf receptor; AM, acetoxymethyl ester; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; 2-APB, 2-aminoethoxy-diphenylborate; N-Rh-Pe, N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine; PBS, phosphate-buffered saline; PM, plasma membrane, AchE, acetylcholinesterase; TG, thapsigargin; IP3, inositol 1,4,5-triphosphate. are endocytic organelles that contain small internal vesicles generated from inward budding of the limiting membrane. In antigen-presenting cells, the fusion of these MVBs with the plasma membrane leads to the release of internal vesicles into the extracellular space (1Clayton A. Court J. Navabi H. Adams M. Mason M.D. Hobot J.A. Newman G.R. Jasani B. J. Immunol. Methods. 2001; 247: 163-174Crossref PubMed Scopus (425) Google Scholar). The released vesicles, termed exosomes (for a review see Refs. 2Stoorvogel W. Kleijmeer M.J. Geuze H.J. Raposo G. Traffic. 2002; 3: 321-330Crossref PubMed Scopus (659) Google Scholar and 3Théry C. Zitvogel L. Amigorena S. Nat. Immunol. 2002; 2: 569-579Crossref Scopus (3835) Google Scholar), were initially described in reticulocyte maturation, where their function was to discard plasma membrane proteins that were no longer necessary, such as the transferrin receptor (4Johnstone R.M. Mathew A. Mason A.B. Teng K. J. Cell. Physiol. 1991; 147: 27-33Crossref PubMed Scopus (214) Google Scholar, 5Harding C. Heuser J. Stahl P. J. Cell Biol. 1983; 97: 329-339Crossref PubMed Scopus (1140) Google Scholar, 6Harding C. Heuser J. Stahl P. Eur. J. Cell Biol. 1984; 35: 256-263PubMed Google Scholar). Although other plasma membrane proteins (e.g. acetylcholinesterase) are secreted via exosomes, these small vesicles are devoid of both cytosolic proteins and proteins associated with other intracellular organelles, indicating that only a select group of macromolecules is shed via this pathway. Exosomes are also secreted by other cell types such as activated platelets, which may function in signaling/adhesion, thus having a role at sites of vascular injury (7Heijnen H.F. Schiel A.E. Fijnheer R. Geuze H.J. Sixma J.J. Blood. 1999; 94: 3791-3799Crossref PubMed Google Scholar, 8Denzer K. Kleijmeer M.J. Heijnen H.F. Stoorvogel W. Geuze H.J. J. Cell Sci. 2000; 113: 3365-3374Crossref PubMed Google Scholar). Exosomes from cytotoxic T cells and B lymphocytes may be involved in targeting molecules for cell death (9Peters P.J. Geuze H.J. Van der Donk H.A. Slot J.W. Griffith J.M. Stam N.J. Clevers H.C. Borst J. Eur. J. Immunol. 1989; 19: 1469-1475Crossref PubMed Scopus (190) Google Scholar) or antigen presentation (10Raposo G. Nijman H.W. Stoorvogel W. Liejendekker R. Harding C.V. Melief C.J. Geuze H.J. J. Exp. Med. 1996; 183: 1161-1172Crossref PubMed Scopus (2517) Google Scholar, 11Zitvogel L. Regnault A. Lozier A. Wolfers J. Flament C. Tenza D. Ricciardi-Castagnoli P. Raposo G. Amigorena S. Nat. Med. 1998; 4: 594-600Crossref PubMed Scopus (1704) Google Scholar). Despite the diverse extracellular functions that are carried out by exosomes, very little is known about the molecular machinery involved in either the formation of the MVBs or in the exosome secretory process. We have recently shown that in K562 cells, a human erythroleukemia cell line, overexpression of Rab11 regulates the exosome pathway (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). Interestingly, treatment of green fluorescent protein-Rab11-transfected cells with the ionophore monensin (MON) generated large MVBs decorated with Rab11 and labeled with a fluorescent lipid that accumulates in exosomes. MON, a membrane-permeable Na+ ionophore that mediates an antiporter activity exchanging Na+ ions with H+ ions (13Pressman B.C. Ann. Rev. Biochem. 1976; 45: 501-530Crossref PubMed Scopus (1457) Google Scholar), acts on acidic intracellular organelles such as endosomes and lysosomes, causing swelling of these vesicles. MON is also known to induce Ca2+ entry by reversed activity of the Na+/Ca2+ exchanger (14Nassar-Gentina V. Rojas E. Luxoro M. Cell Calcium. 1994; 16: 475-480Crossref PubMed Scopus (13) Google Scholar, 15Dömötör E. Abbott N.J. Adam-Vizi V. J. Physiol. 1999; 515: 147-155Crossref PubMed Scopus (45) Google Scholar, 16Wang X.D. Kiang J.G. Scheibel L.W. Smallridge R.C. J. Investig. Med. 1999; 47: 388-396PubMed Google Scholar). A rise in intracellular Ca2+ concentration, a universal intracellular signal (for a review see Refs. 17Cullen P.J. Lockyer P.J. Nat. Rev. Mol. Cell Biol. 2002; 3: 339-348Crossref PubMed Scopus (304) Google Scholar and 18Carafoli E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1115-1122Crossref PubMed Scopus (678) Google Scholar), is necessary to induce regulated secretion in most cell types (reviewed in Refs.19Gerber S.H. Sudhof T.C. Diabetes. 2002; 51: S3-S11Crossref PubMed Google Scholar and 20Wasle B. Edwardson J.M. Cell. Signal. 2002; 14: 191-197Crossref PubMed Scopus (49) Google Scholar). During regulated exocytosis, the membrane of a secretory vesicle fuses with the plasma membrane in a tightly controlled Ca2+-triggered reaction. In endocrine cells, secretory granules contain large amounts of Ca2+ ions, and it has been suggested that the high intragranular Ca2+ concentration is needed to sustain optimal exocytosis (21Scheenen W.J. Wollheim C.B. Pozzan T. Fasolato C. J. Biol. Chem. 1998; 273: 19002-19008Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Because MON generates large MVBs in K562 cells, the aim of the present study was to MON exosome release and Ca2+ is involved in this process. Our results indicate that both MON treatment and a rise in intracellular Ca2+ markedly exosome secretion. Furthermore, the MON-stimulated exosome release was a Ca2+-dependent process. Interestingly, we have also observed that MON the of Ca2+ in the MVBs, suggesting that Ca2+ might be involved in the secretory To a physiological signal might the Ca2+-dependent exosome cells were with transferrin Our results indicate that Tf stimulates exosome release in a Ca2+-dependent cell and were from ester and were from Fluo3-AM, and were from N-(lissamine rhodamine B was from and were from and were from other were from or Cell a human erythroleukemia cell line, was in with and Exosome were from of K562 The were on at for to the cells, and at for to the Exosomes were from the by at for The exosome was in a large of and in of of of released exosomes was by the activity of an that is to these vesicles (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). activity was a described J. 16: PubMed Scopus Google Scholar). of the exosome were in of and with and in a of The was carried out in at and the in at was The the activity at of an exosomes were by the of the by of the were in for at on and to an membrane. The were for in and and with with or with were with and The were an enhanced and by MVBs with the and for fluorescent was into the plasma membrane as described J. M. G. D. Eur. J. Cell Biol. Google Scholar). an of the in was and in was with a into The was to the cells, which were for at this the was and the cells were with to the of and labeled cells were for as described and with were on and by In the cells were with by for at labeling with the fluorescent lipid. were observed these cells were an with the to and to were with a and the were with a and with the of were in the presence of for at were to the extracellular and in cells were from were in the of MON or by MON were in a the of MVBs and Exosome cells are human cells that exosomes R.M. J. Cell. Physiol. 1996; PubMed Scopus Google Scholar), the small internal vesicles released into the extracellular by fusion of MVBs with the plasma membrane has been shown by that treatment of K562 cells with the ionophore MON the formation of MVBs J. Biol. Chem. 1984; Full Text PDF PubMed Google Scholar, A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). the by which these large MVBs are formed and the effect of MON on exosome release have been To into these MVBs in K562 cells were labeled with the fluorescent lipid and have that this lipid is via and to the accumulates in exosomes that are secreted into the extracellular (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar, M. P. D. J. Cell Sci. PubMed Google Scholar). The lipid was to the at and cells were and at for in the or the presence of shown in MON treatment caused the formation of large MVBs labeled by the fluorescent lipid. In a of the MVBs formed is with the internal vesicles labeled with the fluorescent lipid Because fusion of the MVBs with the results in the release of exosomes, we the effect of MON on the release of exosomes from K562 cells. Exosomes are in proteins such as the transferrin receptor and R.M. A. Teng K. Blood. 1989; PubMed Google Scholar). exosomes were in the by the activity of the of and was determined by as described (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). Exosomes were from the extracellular with of MON and by the of the proteins and by shown in MON a increase in exosome release in a A increase was observed by in the the activity of which was at MON in cell were observed as by for which a concentration of MON was used in the of the this concentration, cells were also for by the with of was observed In the of exosomes released was also by the fluorescent lipid this lipid accumulates in intracellular vesicles that are secreted into the extracellular as exosomes. MON also the release of exosomes labeled with the fluorescent lipid the results indicate that MON only generates large MVBs also the secretion of the internal vesicles termed exosomes. A in the Exosome has been shown that MON, a Na+ increase cytosolic Ca2+ by the Na+/Ca2+ (14Nassar-Gentina V. Rojas E. Luxoro M. Cell Calcium. 1994; 16: 475-480Crossref PubMed Scopus (13) Google Scholar, 15Dömötör E. Abbott N.J. Adam-Vizi V. J. Physiol. 1999; 515: 147-155Crossref PubMed Scopus (45) Google Scholar, 16Wang X.D. Kiang J.G. Scheibel L.W. Smallridge R.C. J. Investig. Med. 1999; 47: 388-396PubMed Google Scholar). to in our the enhanced exosome release by MON was to an increase in intracellular Ca2+, we MON the intracellular Ca2+ concentration in K562 cells. this cells were for at with the intracellular Ca2+ concentration was by for of the of that was an Ca2+ and a rise in intracellular Ca2+ that was the The Ca2+ rise was by the of the intracellular Ca2+ chelator Interestingly, in the presence of the extracellular Ca2+ chelator MON the which was to Ca2+ release from intracellular no increase was indicating that the is a of Ca2+ from the extracellular The results that the increase in exosome release might be to a Ca2+-dependent To this we the MON effect on exosome release also be by Ca2+ To the Ca2+ present in the extracellular cells were for in the presence of these the Ca2+ concentration was as with the was used to intracellular Ca2+, this is a membrane-permeable that Ca2+. The released exosomes were from the and by activity as shown in both and the release of exosomes. the increase was completely by the Ca2+ and no were observed both were The that Ca2+ from the extracellular and also from intracellular is for the exosome secretion. Ca2+ in exosome release was the Ca2+ ionophore shown in with the Ca2+ ionophore stimulated exosome secretion to a as were observed both were the secretory effect of the Ca2+ ionophore was by the or is known that MON acts on acidic by the vesicle in a Ca2+ into the K. J. J. J. Cell Sci. 2001; 115: Google Scholar). We the effect of known to the of the and the R. T. A. 2001; PubMed Scopus Google Scholar). has shown that these may also intracellular Ca2+ from acidic Biochem. 1998; PubMed Scopus Google Scholar, R. Cell Calcium. 2001; PubMed Scopus Google Scholar). We have that intracellular Ca2+ in a to MON shown in stimulated the release of exosomes, to a were observed by the of with also the release of exosomes and were by extracellular Ca2+ with the chelator indicating that these via a a by indicate the of intracellular Ca2+ S. S. J. R. Biochem. J. 1996; PubMed Scopus Google for a review see Refs. A. A. Cell Calcium. 1996; 19: PubMed Scopus Google Scholar and R. 2001; PubMed Scopus Google is a that accumulates in the where cytosolic the the fluorescent membrane it has been shown used at of this is of also in intracellular and be used as an for intracellular Ca2+ A. P. B. Physiol. Rev. 1999; PubMed Scopus Google Scholar). were with for at to the MVBs were labeled with the fluorescent lipid as and the cells were with the for at shown in the large MVBs by MON treatment were labeled by Fluo3-AM, indicating that Ca2+ accumulates in these intracellular Ca2+ was also present in the large MVBs formed by The presence of the MVBs calcium in both the of the MVBs was markedly indicating that a is involved in the of the MVBs formed by MON or by and a in the of is that a of the present in the of the P.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), by a Ca2+ from other Ca2+ as as from the extracellular leads to a and increase in the concentration of treatment of K562 with this stimulated exosome secretion in a as MON, and this effect was also by a role for Ca2+ in the exosome secretory pathway and the of a Ca2+ in the process. Because stimulated exosome we were in the large MVBs by MON were also formed by treatment with shown in generated large MVBs the formation of the structures that were with that an increase in cytosolic Ca2+ is by to the MVBs, to exosome secretion. The a role in the of Ca2+ from intracellular (for a review see Refs. Cell. Full Text PDF PubMed Scopus Google Scholar and T. 2001; PubMed Scopus Google Scholar). The for the inositol a of Ca2+ for the of intracellular Ca2+ The increase in the of in the of Ca2+ present in the and the release of Ca2+ into the A. 1991; 51: PubMed Scopus Google Scholar). To this of might be involved in the exosome cells were with 2-APB, a membrane-permeable of Ca2+ that exosome results were with a of indicating that a Ca2+ rise by the of an receptor is critical for the MON-stimulated exosome the formation of the large MVBs by MON was completely by was a in the of the MVBs with the vesicles generated by MON in the of in the cells was the of the to vesicles that the release of Ca2+ via the Ca2+ at in to the of the The of the MVBs with by results indicate that Ca2+ is for of the MVBs, these structures are formed in the presence of Ca2+ to in the the of the large MVBs was only by that of Ca2+ is involved in the Ca2+ rise and the of Ca2+ in the is known that the by MON cytosolic Ca2+ by the Na+/Ca2+ the activity of a Na+/Ca2+ exchanger to be critical for the MON we an of the and Na+/Ca2+ shown in the exosome release and also completely the MON-stimulated secretion of exosomes. the formation of the MVBs generated by was completely by In the was by an of a Ca2+ results are with the that of a Na+/Ca2+ exchanger is a for the Ca2+ rise in the that leads to exosome secretion and the formation of the MVBs with Ca2+ generated by Exosome in a the results indicate that Ca2+ is a in the exosome release process. we were in a physiological stimulus might also exosome secretion in a Ca2+-dependent K562 is a human erythroleukemia cell line that high of TfR, it has been shown that of Tf to receptor intracellular Ca2+ concentration J. V. J. A. Eur. J. Biochem. PubMed Scopus Google Scholar), Tf might be a for exosome secretion. we Tf intracellular Ca2+ in K562 cells. this cells were with as described and human Tf was to the described for other cell types J. V. J. A. Eur. J. Biochem. PubMed Scopus Google Scholar), Tf an increase in Ca2+ that was for the We Tf to the exosome secretion. were for in the presence of human Tf, and exosomes were from the as described shown in Tf stimulated exosome an effect that was by or indicating that was a Ca2+-dependent process. Furthermore, the exosome release was also by 2-APB, suggesting that Ca2+ in this process. is in with a that the of to cells the of C.B. C. J.M. J. 2002; PubMed Scopus Google Scholar). In this study we have shown that the ionophore MON induces the formation of large MVBs and stimulates the release of the internal vesicles exosomes. has been shown that MON induces secretion from cells (14Nassar-Gentina V. Rojas E. Luxoro M. Cell Calcium. 1994; 16: 475-480Crossref PubMed Scopus (13) Google Scholar, A. H. Biochem. 35: PubMed Scopus Google Scholar). The release of regulated secretory granules is known to be we present that the MON-stimulated exosome secretion in K562 cells is a The application of MON generated a of Ca2+ that was on both an extracellular and intracellular Ca2+ The role for Ca2+ on exosome release was by the of the Ca2+ ionophore results were with known to the of such as and an of the stimulated exosome release via a Ca2+-dependent the effect was by We have that intracellular Ca2+ in a as are in with indicating that a Ca2+ release in the Biochem. 1998; PubMed Scopus Google Scholar). it has been shown that cytosolic Ca2+ by intracellular Ca2+ in cells R. Cell Calcium. 2001; PubMed Scopus Google Scholar). our results indicate that Ca2+ is a in the exosome release process. We that this is a on their exosomes play in physiological K. Kleijmeer M.J. Heijnen H.F. Stoorvogel W. Geuze H.J. J. Cell Sci. 2000; 113: 3365-3374Crossref PubMed Google Scholar). activated release exosomes at sites of vascular injury where may have a function (7Heijnen H.F. Schiel A.E. Fijnheer R. Geuze H.J. Sixma J.J. Blood. 1999; 94: 3791-3799Crossref PubMed Google Scholar). cells also exosomes that molecules as for our that Ca2+ regulates exosome release that a signal is involved in the of cells to release these small vesicles at the The of MON to that Ca2+ an role in exosome secretion. has been that MON acts on acidic intracellular organelles such as endosomes and lysosomes, exchanging H+ for Na+ and causing swelling of these vesicles by Cell. 1983; Full Text PDF PubMed Scopus Google Scholar). it be that a might be involved in the of the large MVBs formed MON our results indicate that the formation of the endosomes is a this was completely by the Ca2+ chelator is to that the formation of these endosomes is only to swelling of the vesicles also the of from other implying fusion is that intracellular on Ca2+ A. Cell Biol. 1999; PubMed Scopus Google Scholar) it is that Ca2+ might be for the fusion involved in the of the are to this MON, as a Na+ a of Na+ the cells of H+ to the extracellular The increase in intracellular Na+ the Na+/Ca2+ exchanger in a to an increase in cytosolic Ca2+ X.D. Kiang J.G. Scheibel L.W. Smallridge R.C. J. Investig. Med. 1999; 47: 388-396PubMed Google Scholar). Our are in with a requirement for an of the were completely by an of the and Na+/Ca2+ Our are also with the that Na+ entry by MON the of IP3, which in releases Ca2+ from intracellular the of these might the of Ca2+ at the plasma membrane that induces a Ca2+ rise with the requirement for extracellular Ca2+ in our in this indicate that are involved in MON-stimulated exosome indicating that a Ca2+ rise by the of is critical for this the formation of the MVBs was only and completely by of the suggesting that an might be involved in the of these large Interestingly, we have observed that the large MVBs generated by are with Ca2+. is that the MON-stimulated activity might be at the plasma membrane, it is also that a effect on intracellular it has been shown that MON induces the secretion of by the membrane C. J. Biol. 1991; PubMed Scopus Google Scholar). and K. J. J. J. Cell Sci. 2001; 115: Google Scholar) have that contain high concentration of Ca2+ and function as an intracellular Ca2+ have shown that changes in in the of Ca2+ out of into the via calcium or Our results indicate a Ca2+ is involved in the release of exosomes, of this either the formation of the MVBs or the of Ca2+ the large it is that of Ca2+ present in intracellular might be for the a Ca2+ present in A. G. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) to the plasma membrane Ca2+ is A Ca2+ in the of J. Physiol. Cell Physiol. 2000; PubMed Google Scholar) with from the and the Ca2+ has also been secretory pathway to be a to the and is to be for the function of the secretory pathway. Interestingly, we have recently shown that MVBs in K562 cells are at in by membrane from (12Savina A. Vidal M. Colombo M.I. J. Cell Sci. 2002; 115: 2505-2515Crossref PubMed Google Scholar). Furthermore, it has been shown that endocytic vesicles from a Ca2+ that Ca2+ into the lumen of the vesicles M. B. J. J. Biochem. Mol. Biol. 35: Google Scholar). it be that a Ca2+ is involved in the formation of the MVBs and the of the Ca2+. possibility is that the Ca2+ present the MVBs is a critical role in the exosome secretory process. A role for Ca2+ in secretory and intracellular fusion has been Fusion of endosomes to the release of Ca2+ for fusion to S. M.J. J. Cell Sci. 2001; PubMed Google Scholar). in cells it has been shown that Ca2+ from granules exocytosis (21Scheenen W.J. Wollheim C.B. Pozzan T. Fasolato C. J. Biol. Chem. 1998; 273: 19002-19008Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). the presence of on and secretory granules has been suggesting that these organelles a Ca2+ the secretory H. Proc. Natl. Acad. Sci. U. S. A. 1994; PubMed Scopus Google Scholar). we it is known that MON the the release of Ca2+. it is that the release of Ca2+ from the MVBs at the may play an role in the fusion our results indicate that Ca2+ is a critical in the MON-stimulated exosome are to the Ca2+ in this process. we have indicating that Tf intracellular calcium in K562 cells and stimulated exosome release in a Ca2+-dependent manner, suggesting that the secretion of exosomes is physiological Our results are with that the of transferrin to receptor intracellular Ca2+ and stimulates receptor in cells J. V. J. A. Eur. J. Biochem. PubMed Scopus Google Scholar). transferrin was stimulated by Ca2+ in cells Traffic. 2002; 3: PubMed Scopus (13) Google Scholar). Our results that a of the machinery involved in exosome secretion is regulated by Ca2+. such as and have been in where function as Ca2+ Biochem. J. 2002; PubMed Google Scholar). Tf, by increasing intracellular Ca2+, may critical of the Interestingly, in a it has been shown that the of to receptor via a the of and proteins known to be involved in C.B. C. J.M. J. 2002; PubMed Scopus Google Scholar). In the to function as a signal that only also via the exosome and Ca2+ is of the of this pathway by Tf is to that exosomes released to from cells are the of the R.M. J. Cell. Physiol. 1996; PubMed Scopus Google Scholar). have been in K. J. PubMed Scopus Google Scholar). the physiological role of the J. J. 2002; PubMed Scopus Google Scholar) has been our that Tf stimulates exosome release as a the of TfR, to that this is a to the of We Amigorena and for critical of this We also for

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.