Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for serious tropical diseases in humans. The parasites are transmitted by insects and, consequently, have a complicated life cycle during which they undergo dramatic morphological and metabolic transformations to adapt to the different environments. Autophagy plays a major role during these transformations. Since inhibition of autophagy affects the transformation, survival and/or virulence of the parasites, the ATGs offer promise for development of drugs against tropical diseases. Furthermore, various trypanocidal drugs have been shown to trigger autophagy-like processes in the parasites. It is inferred that autophagy is used by the parasites in an—not always successful—attempt to cope with the stress caused by the toxic compounds.Autophagy is a ubiquitous eukaryotic process that also occurs in trypanosomatid parasites, protist organisms belonging to the supergroup Excavata, distinct from the supergroup Opistokontha that includes mammals and fungi. Half of the known yeast and mammalian AuTophaGy (ATG) proteins were detected in trypanosomatids, although with low sequence conservation. Trypanosomatids such as  Trypanosomatidae is a family of protists that belong to the major clade, Kinetoplastea . These aTrypanosoma and Leishmania stand out, responsible for diverse clinical manifestations including major human diseases, such as Chagas disease, sleeping sickness and leishmaniasis. Chagas disease consists of a chronic and systemic infection caused by Trypanosoma cruzi. The disease affects about eight million people in Latin America, of whom 30%–40% either have or will develop cardiomyopathy, digestive megasyndromes, or both. Nowadays it is also becoming an emerging health problem in non-endemic areas because of increasing population movements . O. OT. bruviig bug B 24]. O. OT. brund flies C 25]..T. bruceDuring their developmental cycles, trypanosomatids face very different conditions of pH, temperature, nutrients and oxygen supply. Consequently, the parasites’ metabolism differs in the different life-cycle stages, requiring an important metabolic reprogramming. In that aspect, autophagy is expected to play an important role.Saccharomyces cerevisiae. More than 30 ATG (AuTophaGy-related) genes have been identified and, for many of them, molecular and functional studies have been performed, facilitated by the easy manipulation of these organisms [S. cerevisiae and Pichia pastoris, where in nutrient-rich conditions specific hydrolytic enzymes are routed, by use of dedicated vesicles, to their final destination, the vacuole [The word autophagy was coined by Christian de Duve in 1963, to define the process by which the cell’s can degrade its own proteins . Previourganisms ,28,29. Arganisms ,32. Macr vacuole . The bes vacuole , or mito vacuole , pieceme vacuole  among ot vacuole ,38,39,40Another mechanism participating in the recycling of intracellular proteins is the proteasome system. This system, conserved in all eukaryotes, drives mainly misfolded and aggregated cytosolic proteins marked with one or several ubiquitin moieties to a multiprotein complex, the proteasome, where proteolysis occurs. Protein modification by ubiquitin does however not serve exclusively for targeting proteins for proteasomal degradation. This signal also participates in cellular trafficking, immune response, DNA repair and chromatin remodeling . This moIn the last decade it has been shown that lysosomes and autophagy are present in a large variety of eukaryotes, including trypanosomatids ,44,45. Ide novo formation of the double-layered isolation membrane at the vesicle nucleation step. The vesicle expansion step is next, where different ATGs participate to bring lipids for increasing the membrane’s size and curvature. The closure of the isolation membrane causing the separation of its contents from the cytosol comprises the vesicle completion step. The newly formed vesicle is the so-called autophagosome. Next, the fusion of the outer membrane of the autophagosome with the lysosome/vesicle is followed by the degradation of the single membrane autophagic body and release of its contents into the lysosome/vesicle.Irrespective of the conditions that cause the need for degradation of cytoplasmic components, the autophagy pathway in any cell follows a similar series of steps  from itsThe complex comprising ATG1-ATG13-ATG17 in yeasts and ULK1-ATG13-FIP200-ATG101 in mammalian cells is involved in autophagy induction . It is uS. cerevisiae has identified in trypanosomatids homologues for about 20 of these gene products that participate in each of the steps of the pathway in yeast [The trypanosomatids genome databases and the range of ATGs identified from yeast species have been used to predict if the parasites possess a conserved autophagy pathway. An initial BLAST search using 40 ATGs and other proteins involved in autophagy from in yeast . While ain yeast , where min yeast . A summaT. brucei possesses one copy for TOR1 and TOR2 as well as a TOR-like 1 and a TOR-like 2 gene [L. major has a TOR-1, a TOR-2 and a TOR-3 [T. brucei. T. cruzi has a TOR1 and a TOR2.One obstacle for the characterisation of the complex involved in autophagy induction in the parasites is the fact that trypanosomatids possess too many possible homologues for the kinase ATG1. Upstream acting TOR1 and TOR2 homologues were readily identified in all three trypanosomatids. e 2 gene , while L a TOR-3 , with thThe class III phosphatidylinositol 3-kinase (PI3K) VPS34 and its interacting partners, that participate in the vesicle nucleation step (ATG6/VPS30 and VPS15), possess homologues in the trypanosomatid genomes, however ATG14 and VPS38, responsible for the specificity of the complex between autophagy and ‘vacuolar protein sorting’, seem to be absent.T. brucei, two for T. cruzi and, surprisingly, four families comprising together 25 genes for Leishmania major. T. brucei has two very similar ATG8s (ATG8.1 and ATG8.2) and a more distant ATG8.3. T. cruzi has a ‘true’ ATG8.1 and an ATG8.2 that does not seem to participate in autophagy [For the autophagosome marker ATG8, more than one gene were identified for each of the trypanosomatids: three for utophagy .L. major [T. cruzi [Unlike yeast, where one ATG8 and one ATG4 are found, and more similar to mammals having several homologues of ATG8 with apparently different roles in the cell, trypanosomatids possess not only multiple ATG8 homologues but also two paralogues for ATG4. Different specificities and efficiencies have been described for L. major  and T. cT. cruzi  ATG4s  are often found to be exclusive to one species or its closest relatives [Moreover, homologues for the genes of general autophagy-related proteins ATG13, ATG14, ATG17, ATG22, ATG23 and ATG27, and of the Cvt pathway-specific ATG11, ATG19, ATG20 and ATG21 were not found in the trypanosomatids, nor genes for the yeast mitophagy-specific UTH1 and AUP1 and pexophagy-specific ATG11, ATG25, ATG28 and ATG30. While the Cvt pathway seems restricted to fungi, the absence of several specific autophagy homologues in the trypanosomatid genomes should not be taken as an indication that there is no specific autophagy taking place in these cells, as it is known from studies with yeasts that specific-autophagy genes such as ATG28 and ATG30 in elatives ,60, withelatives , other ielatives . Surely,T. brucei taken by Vickerman and colleagues in the 1970s [As described in he 1970s . HoweverT. brucei lives in an environment where glucose is present at a reasonably constant level. Consequently, and because the parasite’s single mitochondrion is highly repressed at this stage, bloodstream forms obtain all their energy by generating ATP via glycolysis. The majority of the enzymes of the glycolytic pathway are uniquely sequestered in peroxisome-like organelles which were hence called glycosomes [The bloodstream form of ycosomes ,64. Thisycosomes ,66. Thisycosomes ,11,67. Bycosomes . In agreycosomes ,9.T. brucei, which seems reminiscent of the situation observed for specific autophagy of peroxisomes (pexophagy) in methylotrophic yeasts [in vitro cultured parasites. In this way, ‘old’, redundant glycosomes are degraded while new organelles become synthesized, containing an enzyme repertoire better adapted to the lifestyle of the new form of the parasite. The fact that the lysosome shows a transient but dramatic increase in size during the differentiation process could suggest that macropexophagy would be involved in the formation of autophagosomes. However, glycosomes seem to accumulate at the lysosome and become surrounded by it, suggesting quite the opposite, a process similar to micropexophagy [S. cerevisiae are present in T. brucei [Leishmania species, where only macroautophagy has been experimentally observed so far, as it will be described below [Strong indications have been obtained that autophagy participates in this remodeling of the number, size and enzymatic contents of the glycosomes during the development of the parasite. Electron microscopy pictures confirm that glycosomes can be sequestered within double-membranes of autophagosome-like structures . Moreovec yeasts . A tendeexophagy . This la. brucei . This seed below ,71.TbATG8.1 and TbATG8.2, and one called TbATG8.3. This latter one is, based on phylogenetic criteria, a typical ATG8, but its gene is syntenic to that of a protein which in L. major has been shown to exert the ATG12 function (see below). Punctate structures proposed to be autophagosomes were detected by the expression of a fusion construct of yellow-fluorescent protein and TbATG8.2 in procyclic cells exposed to endoplasmic reticulum (ER) stress by DTT treatment. However, these autophagosomes appeared in cells that also expressed apoptotic markers, resembling the apoptosis process that takes place in mammalian cells under persistent ER stress [TbATG8.2 has been obtained, showing the classic ubiquitin fold [TbATG8.2 is the functional homologue of the human ATG8/LC3. Upon starvation or upon induction of differentiation, both TbATG8.1 and TbATG8.2 concentrate in T. brucei into punctate structures reminiscent of autophagosomes [TbATG8.1 and TbATG8.2 appear not to be fully equivalent. Depletion of TbATG8.1 does not cause detectable effects on TbATG8.2 recruitment to autophagosomes, whereas TbAtg8.2 depletion significantly reduces the autophagosome relocation of TbAtg8.1. Furthermore, Li et al. [TbATG8.1 and TbATG 8.2, individually or together, promotes cell survival under starvation conditions. Unexpectedly, their results seem to indicate that autophagy in trypanosomes is detrimental when the cells are brought under starvation conditions. T. brucei also contains two ATG4 homologues, TbATG4.1 and TbATG4.2. When docking TbATG8.2 to a homology model of TbATG4.1 (the one with higher sequence identity with human ATG4), the overall structure seems very much alike and the necessary catalytic triad (C-D-H) is well conserved in TbATG4.1, strongly suggesting conservation of function [The typical marker used for the study of autophagy in many organisms is ATG8. As already mentioned, African trypanosomes possess three ATG8 isoforms: two which are more closely related to human ATG8/LC3, R stress . Furthertin fold . This, tagosomes ,73. Howei et al.  describefunction .target of the antibiotic rapamycin. There are two different TOR-containing complexes in all the eukaryotes studied, with capabilities of governing spatial and temporal cell growth separately. Generally, complex 1 (TORC1) controls temporal aspects of cell growth through processes such as ribosome biogenesis, transcription, translation and repression of autophagy, while TORC2 controls spatial aspects of cell growth by actin cytoskeleton remodeling [T. brucei (TbTOR1 and TbTOR2), where they bind to and control signaling through TbTORC1 and TbTORC2, respectively [TbTOR1 is mainly located in the nucleus, whereas TbTOR2 is excluded from the nucleus and is associated with the ER and the mitochondrion. In addition, trypanosomatids present two more TOR-related proteins, TbTOR-like 1 and TbTOR-like 2, that do not seem to form part of the TOR complexes. As in other eukaryotes, TbTOR2 seems to regulate actin polarization towards the endocytic pathway, playing a crucial role in endocytosis and cytokinesis, while the TbTOR1 function is to promote cell growth and proliferation by positively regulating protein synthesis, cell size, cell cycle progression and RNA polymerase I localization to the nucleolus. However, when bloodstream forms of T. brucei were treated with rapamycin, there was a pronounced reduction of cell proliferation but, contrary to what occurs in other eukaryotes, the effect was caused by inhibition of TORC2, rather than TORC1. Used in the nanomolar range, rapamycin inhibits TbTORC2, producing actin depolarization which interferes with endocytosis and cytokinesis. Moreover, a rapamycin-FKBP12 complex (FKBP12 is the mediator that lets rapamycin to bind TOR) does not bind to the recombinant TbTOR1 rapamycin-binding domain, while it does to TbTOR2 both in vitro and in vivo. Taken together, these experiments suggest that rapamycin cannot be used for triggering the autophagic process in T. brucei, because TbTOR1 seems to be rapamycin-insensitive [T. brucei (apparently different from apoptosis and necrosis) with the formation of structures that are reminiscent to phagophores, autophagosomes and autolysosomes. However, in order to observe this effect, a concentration 10-fold higher than that used to induce autophagy in yeast was required [TbTOR1 knockdown triggered the appearance of autophagic-like vesicles. Its depletion also caused morphological changes such as abnormal appearance of the ER and formation of multi-lamellar membranes, and it conferred partial resistance to stress situations, as it happens in other eukaryotes when TOR1 is inhibited [In higher eukaryotes, the balance between protein synthesis and degradation via autophagy is regulated by TOR, a well-studied kinase that is mostly known, and received its name for being the modeling . In animmodeling ,75. TOR1ectively ,76. As iensitive . Nonetherequired . MoreoveTrypanosoma and Leishmania species were highly susceptible for mammalian mTOR and PI3K inhibitors that are currently in pre-clinical and clinical development for cancer treatment [T. brucei infected animal model. Although TOR as the in vivo target of these compounds in the parasites remains to be proved, the results are highly promising. Moreover, the large sequence difference of the human and trypanosome enzymes offer promise for increasing the selectivity of the inhibitory compounds.The possibility of using trypanosomatid TOR enzymes as drug targets received recently further support by the demonstration that reatment . SeveralT. brucei bloodstream forms (with an EC50 of 1 mM) as a result of cell cycle arrest in the G2/M phase, as well as morphological alterations such as an increase of vesicular structures within the cytosol and the presence of multivesicular bodies, myelin-like structures, autophagy-like vacuoles and a marked disorder of the characteristic mitochondrion structure. Furthermore, Delgado and colleagues reported that some neuropeptides can be endocytosed by T. brucei bloodstream forms, inducing autophagy in the parasites [In several cases it has been shown that the process of autophagy can also be induced in African trypanosomes by the addition of different molecules to the medium. One of these molecules is dihydroxyacetone (DHA) ,80. Whilarasites ,82. The arasites ,82.T. cruzi epimastigotes into metacyclic infective forms that occurs in the insect's rectum may be triggered by the stress of nutrients limitation. The insect’s epimastigote stage contains a lysosome-like organelle designated the reservosome, that stores endocytosed material, lipids and secretory proteins, as well as a major cysteine proteinase, cruzipain [in vitro by inducing starvation of cultured epimastigotes. Cruzipain seems to be the responsible for this proteolysis, as inferred from the fact that cysteine proteinase inhibitors can block the differentiation [It has been described that the process of differentiation of ruzipain . Apparenntiation ,85.T. cruzi has not yet been addressed. Similarly for TcVPS34, whose role in autophagy remains to be elucidated. However, this class III PI3K has been shown to be involved in osmoregulation and receptor-mediated endocytosis [The role played by the TOR kinases in autophagy (or any other pathway) in ocytosis .T. cruzi contains two ATG8 homologues, TcATG8.1 and TcATG8.2. It was observed that differentiating and starved epimastigotes show an intense TcATG8.1 staining. In contrast, almost no signal was found in metacyclic trypomastigotes, and the weakly visible TcATG8.1 co-localized in part with the carboxypeptidase located in reservosomes. This suggests the delivery of the autophagosomal content to the lysosome system, which occurs as one of the final steps of the autophagy process [As mentioned above,  process .T. cruzi also contains two ATG4 isoforms, TcATG4.1 and TcATG4.2. In an in vitro cleavage assay, TcATG4.1 was able to rapidly process both TcATG8.1 and TcATG8.2, while TcATG4.2 showed little activity towards these proteins. However, both TcATG4 genes could complement a yeast ATG4 mutant. On the other hand, only TcATG8.1 could partially replace the function of yeast ATG8, while TcATG8.2 could not. Moreover, only HA-tagged TcATG8.1 localized to large vesicles that were thought to correspond with autophagosomes. This localization appeared to be dependent on the key glycine residue at the C-terminus of the protein, since mislocalization occurred when the residue had been mutated. By contrast, HA-tagged TcATG8.2 only showed a weak punctate localization during starvation, and its function still remains unknown [TcATG8.1 is the functional homologue of yeast and mammalian ATG8. unknown . Taking T. brucei, several molecules have been described to induce autophagy in T. cruzi. Three protein kinase inhibitors, staurosporine (a serine/threonine kinase inhibitor), genistein (a tyrosine kinase inhibitor) and the PI3K inhibitor wortmannin were able to cause antiproliferative effects in T. cruzi epimastigotes with the production of autophagosomes, among other ultrastructural changes. However, these drugs did not interfere with the division of intracellular amastigotes or with their differentiation to trypomastigotes [Pterodon pubescens, caused the appearance of concentric membrane structures in the cytosol associated to the autophagy process. Propolis, a resinous material collected by honeybees from different plant exudates, led to reservosome disorganization in epimastigotes. Naphthoquinones derived from β-lapachone (obtained of Tabebuia sp. trees) have been shown to cause atypical membrane structures, suggestive of the involvement of autophagy [As in stigotes . Other cstigotes . Other tstigotes . Furtherutophagy . More reutophagy . Finallyutophagy ,91,92. Tutophagy . The deautophagy .Leishmania also face very different conditions during their life cycle. Leishmania promastigotes living in the alimentary tract of a sand fly are exposed to a temperature of around 26 °C and rather basic conditions, whereas the amastigotes present within the phagolysosome of mammalian macrophages are exposed to a higher temperature, an acidic pH, low levels of oxygen and different nutrimental conditions [As trypanosomes, the multiple species that belong to the genus nditions . The comnditions .T. brucei, the available data suggest that the metabolic reprogramming required during differentiation of promastigotes to amastigotes does not involve increased glycosome turnover by pexophagy. The altered metabolic fluxes seem rather the result from different nutrient availability and posttranslational regulation of changes in enzyme activities, than from the introduction of major changes in the metabolic enzyme repertoire [Leishmania, pexophagy may be more involved in an adjustment of the number of glycosomes, associated with the dramatic decrease of cell size during this differentiation step, instead of a switch of the glycosomal enzyme content [However, and contrary to what has been described for pertoire ,97. In L content ,99.Leishmania species, the lysosome presents multiple shapes which reflect the distinct functions in the various developmental forms [Leishmania genome [L. major VPS4 gene resulted in an accumulation of cytosolic autophagosomes that could not be processed further in the MVT-lysosome. VPS4 is an ATPase involved in the endosomal system and in the formation of late endosomes with a multivesicular aspect called multivesicular bodies (MVB). As a consequence of VPS4 inactivation, the ability of the parasites to survive under nutrient deprivation conditions was affected and they were unable to differentiate into metacyclic promastigotes. In addition, a mutant of the ATG4.2 cysteine peptidase from L. major was also found to be defective in the ability to differentiate, supporting a role of autophagy during the differentiation process. Although in this mutant GFP-ATG8 puncta were still present during starvation, highly increased levels of the lipidated form of ATG8 were observed [in vitro experiments showed that LmATG4.1 presents higher proteolytic activity towards LmATG8 than LmATG4.2 does [LmATG4.2 mutation affects the release of LmATG8 from PE at the autophagosomal membrane, rather than the activation of LmATG8 prior to its conjugation to PE.On the other hand, it has been shown that protein turnover by macroautophagy is essential for metacyclogenesis (the differentiation of procyclic promastigotes into highly infective metacyclic promastigotes) and also for the differentiation of metacyclic promastigotes into amastigotes, as reviewed by Besteiro and colleagues . In the al forms . While pa genome . Moreoveobserved . Moreove4.2 does . Taken tLeishmania: ATG8, ATG8.A, ATG8.B and ATG8.C. Together, the different families are able to fulfill the action of the unique ATG8 from yeast [LmATG8 shows a high similarity to yeast and mammalian ATG8, and has been successfully used to label autophagosomes [LmATG8.A also appears to localize to autophagosomes in promastigotes, but participating only in starvation-induced autophagy, while LmATG8.B and LmATG8.C seem not to be involved in autophagy [L. major also presents an ATG8-like protein with a C-terminal extension after the key glycine residue that is essential for conjugation to ATG5. Although this protein clusters with ATG8s in a phylogenetic analysis, it was named LmATG12 because it contains a 58-residue insertion that makes it quite similar to yeast and mammal ATG12, and indeed behaved as an ATG12 in yeast complementation studies. LmATG4 proteins are unable to cleave the C-terminal extension from LmATG12, but since only a mutated LmATG12 with a free C-terminal glycine can rescue a yeast ATG12 null mutant, it is thought that an alternative peptidase must be present in order to free the glycine residue. L. major homologues of ATG5 and ATG10, despite their weak similarity, could also replace their yeast counterparts [LmATG12 partially co-localizes with GFP-LmATG8 to punctate structures in starving L. major promastigotes. Additionally, Williams and colleagues have recently demonstrated the presence of a functional ATG12-ATG5 conjugation system in L. major, which is required for ATG8-dependent autophagosome formation. A null mutant of ATG5 and in consequence the lack of autophagy, leads to perturbation of the phospholipid balance in the mitochondrion, possibly through ablation of membrane use and conjugation of mitochondrial PE to ATG8 for autophagosome biogenesis, resulting in a dysfunctional mitochondrion with impaired oxidative ability and energy generation that causes a decrease in the virulence of the parasites [As mentioned above, four families of ATG8 were found in om yeast , but onlagosomes . LmATG8.utophagy . Curiousterparts . LmATG12arasites .LmTOR1 and LmTOR2 are essential proteins and have not been characterized yet, however LmTOR3 has been shown to be involved in acidocalcisome biogenesis [L. donovani by interfering with the autophagy pathway. Starvation conditions cause the overexpression of the protein, which leads to an acceleration of autophagy. Consistently, low levels of Ldpkar1 delay the induction of autophagy in the parasite [The autophagy inducing proteins, ogenesis . Recentlparasite .Leishmania that can be related to autophagy. The aziridine-2,3-dicarboxylate-based cysteine cathepsin inhibitor 13b  induced cell death in L. major promastigotes. Accumulation of debris in autophagy-related lysosome-like vacuoles was observed at an early phase of the dying of the parasites, but death seemed to finally occur by an apoptosis-like death mechanism [L. chagasi and L. amazonensis promastigotes treated with yangambin, a lignan obtained from Ocotea duckei, also showed features of both apoptosis and autophagy [Laurencia dendroidea, showed an antiproliferative effect against L. amazonensis promastigotes (at 4 μM) and amastigotes (at 0.45 μM), causing diverse changes including the appearance of concentric membrane structures and formation of membrane structures suggestive of an autophagic process [L. amazonensis parasites treated with Amiodarone, a drug commonly used to treat arrhythmics, presented amongst many other alterations including severe mitochondrial swelling, the formation of large autophagosomes that contained part of the cytoplasm and membrane profiles [L. donovani promastigotes with the appearance of vacuoles that were stained with monodansylcadaverine, a biochemical marker of autophagy [Several compounds have been also shown to cause ultrastructural alterations in echanism . L. chagutophagy .The sesq process . More reprofiles . Antimicutophagy .T. cruzi infects mammalian cells, a signal transduction cascade is activated that leads to the formation of its parasitophorous vacuole, which has been shown to be labeled by the host cell autophagic protein LC3. Furthermore, autolysosomes are recruited to parasite entry sites, and starvation or pharmacological induction of autophagy of mammalian cells significantly enhances the infection, whereas inhibitors of the autophagic pathway reduce the invasion. Moreover, the absence of Atg5 or the reduced expression of Beclin 1, two proteins required at the initial steps of autophagosome formation, limit parasite entry and reduce the association between the parasitophorous vacuole and the classical lysosomal marker Lamp-1 [The experimental evidence for autophagy in trypanosomatids presented above provides us with a quite realistic idea of the multiple roles that autophagy can play in these parasites. As we have seen, one of the most important roles of autophagy is related to the differentiation of the parasites during their life cycle . Autophar Lamp-1 . L. donovani has been shown to result from the cells counteracting the toxic effects of the compound by eliciting initial autophagic features. Indicating that autophagy indeed serves as a survival mechanism in response to cryptolepine treatment in L. donovani promastigotes, is the fact that inhibition of autophagy causes an early increase in the number of cells that die [T. brucei [We have also mentioned several examples of how autophagy has been described to be a mechanism of cell death in trypanosomatids, as it has been previously described for other organisms. In some of these cases, the appearance of autophagosome-like bodies seems to be accompanied by the characteristic cellular integrity that corresponds with an autophagy process, where treated cells present a normal nucleus and show no alteration of the mitochondrion, nor permeabilization of the cellular membranes. However, in many cases the cell phenotype is characterized by a mix of structural changes that could correlate with both autophagy-related and apoptotic-like processes. A new link between autophagy and apoptosis has been recently discovered in mammals, showing that ATG12 can bind to anti-apoptotic Bcl-2 family members to promote apoptosis ,109. Thithat die . Contrar. brucei .T. brucei proteasome is the best-characterized one in any parasitic protist. Relevant for drug discovery is the fact that not only important differences exist between the proteasome components of trypanosomatids and mammalians, but also that the requirement of this complex for the proliferation and differentiation of the parasite has already been established for T. brucei, T. cruzi and L. mexicana [Proteasomes play also a role in protein recycling in addition to autophagy. This protein complex with protease activity that works independently of the lysosome, is conserved in all eukaryotes, including trypanosomatids. The mexicana ,113. Indmexicana .The occurrence of autophagy in trypanosomatid parasites has been convincingly demonstrated, first by bioinformatics approaches, and subsequently experimentally in a variety of molecular and cellular studies. Previously, we have shown, on the basis of genomic searches, that autophagy is a ubiquitous eukaryotic process, because a canonical autophagy machinery is present in representatives of all major eukaryotic lineages thus must have already been present in the common ancestor ,54. Howei.e., turnover of proteins to eliminate damaged cell constituents, such as proteins and organelles. However, a more important role is exerted in the remodeling of the morphology and metabolic capacity of the parasites during the various differentiation processes associated with the successive transitions in their complicated life cycle that involves different hosts and host compartments, exposing the parasites to highly different environments. Since these differentiation processes are essential for the parasites, their autophagy pathways may be a promising drug target against the serious tropical diseases caused by them and for which no adequate therapy is available. The low degree of sequence conservation of ATG proteins reinforces the notion that these proteins might be suitable targets for the development of trypanosomatid versus human protein selective inhibitors. Such compounds might then be used as leads for the development of drugs against diseases such as sleeping sickness, Chagas disease and leishmaniasis. In addition, considerable evidence is available that treatment of the parasites with a large variety of toxic compounds trigger autophagy-like processes. It is inferred that autophagy is used by the parasites in an attempt to cope with the stress caused by the compounds. Often the response is insufficient, and the parasites die. Some authors interpret this as ‘autophagy-related cell death’, but it remains to be established whether it should not rather be considered as ‘cell death despite autophagy’. In the latter case, combination therapy of drugs interfering with both metabolic processes and autophagy in the parasites may have an additive or even synergistic effect.Autophagy in these parasites may—at a basal level—play a similar role as in other eukaryotes, We are not aware of any study on the effects of the currently recommended drugs for Chagas disease, sleeping sickness or visceral leishmaniasis on autophagy in the parasites, or the importance of autophagy in the manner by which the drugs exert their effects. However, it has recently been reported that a drug used for cardiac arrythmia in chronic Chagas patients administered in combination with a promising drug in Phase 2 of clinical trials also for Chagas treatment, induces autophagy and many other morphological defects in the cells. Although autophagy is probably upregulated as a rescue mechanism to protect the cell against the toxic effects of the drugs, the authors conclude that the parasites die as a result of autophagy .Together, the data presented in this paper indicate that further research on autophagy in trypanosomatid parasites may provide important fundamental scientific data about a process that shows considerable differences with those of the well studied yeasts and mammals, and might be highly relevant for developing new chemotherapeutics against serious neglected tropical diseases.


With cases of severe acute respiratory syndrome (SARS) occurring across geographic regions, data collection on the effectiveness of intervention strategies should be standardized to facilitate analysis. We propose a minimum dataset to capture data needed to examine the basic reproduction rate, case status and criteria, symptoms, and outcomes of SARS. First detected in China, confirmed and probable cases of severe acute respiratory syndrome (SARS) have now appeared in at least 30 countries in five continents. SARS is the first new severe infectious disease to occur in the 21st century, and little is known about its epidemiologic features  are collected. Suggested categories for symptoms include fever, myalgia, dyspnea, headache, chills, diarrhea, nausea, sore throat, arthralgia, chest pain, productive cough, nonheadache neurologic symptoms , rhinorrhea or runny nose, vomiting, and abdominal pain. The list of symptom categories can be revised or extended as needed.Outcome variables include hospitalization , treatment status , isolation start date, number of days isolated (a continuous variable), number of days on ventilation or in intensive care (continuous variables), discharge date , death (yes or no), and date of death. Appendix provides an example of Priority group 1 variables  contain the information on the transmission rate of the disease and incubation periods. These variables provide crucial information in determining the basic reproduction number of an infection (defined as the expected number of secondary infectious cases resulting from one primary case in a susceptible population) ,5. This The emergence of a novel disease like SARS, which requires a global public health response to contain its spread, has illustrated the need for collecting effectiveness data in a uniform manner. Given the potential for a large variation in location-specific circumstances, producing a single questionnaire that would be entirely suitable for all locales would be difficult. A 55-year-old man traveled to mainland China from March 5 to March 15. By March 18, the man had a temperature of 38.5°C, cough, and sore throat. He was hospitalized on March 20, and results from radiographic tests indicated pneumonia. The patient was given a course of antimicrobial drugs, and the fever resolved. By March 23, the patient began complaining of chest pains and had difficulty breathing. At this point, SARS was suspected, and the patient was moved into isolation in an intensive care unit the same day and had to be intubated for 5 days. Results from the polymerase chain reaction (PCR) test indicated the presence of the SARS-associated coronavirus. The patient began antiviral treatment and remained isolated in intensive care until April 14. Infection resolved, and the patient was discharged on April 16. The patient had no previous history of serious respiratory illness or any other serious coexisting conditions, and the source of the exposure could not be determined.The 50-year-old wife of patient 1 was exposed to SARS upon her husband's return on March 15. On March 23, a cough developed, and she had shortness of breath and chills. She was hospitalized on March 24 with a temperature of 38.1°C. Radiographic tests indicated evidence of pneumonia. Because SARS was suspected, the patient was placed on antiviral treatment, and SARS was confirmed by PCR testing. Patient 2 was placed in isolation on a medical ward on March 25 and remained there until April 11. She was discharged on April 12. This patient was also diabetic.The 15-year-old daughter of patients 1 and 2 also became exposed to SARS when her father returned on March 15. She has a mild asthma condition. On March 23, she complained of stomach pain and, upon examination, had a temperature of 37.7°C. She was hospitalized on March 25 for observation, but no further symptoms developed. PCR testing returned negative results for the SARS coronavirus. Her fever resolved, and she was released on March 27.The 23-year-old daughter of patients 1 and 2, who lived away the family home, became exposed to SARS through contact with both patients 1 and 2 upon a brief visit (lasting <1 hour) on March 19. By March 27, she had a cough and a temperature of 38.1°C. She contacted her physician and was hospitalized on March 28 for observation and evaluation. Results of radiographic testing did not show pneumonia. She began an antiviral treatment program and began to improve. A PCR test was not completed because her fever resolved, and no other symptoms developed. She was discharged on April 1.Empty table used in Figure 1. Edit 1 Empty table used in Figure 2.


Identification keys are decision trees which require the observation of one or more morphological characters of an organism at each step of the process. While modern digital keys can overcome several constraints of classical paper-printed keys, their performance is not error-free. Moreover, identification cannot be always achieved when a specimen lacks some morphological features . DNA barcoding was proven to have great potential in plant identification, while it can be ineffective with some closely related taxa, in which the relatively brief evolutionary distance did not produce differences in the core-barcode sequences.rbcL and matK), plus the additional trnH-psbA region, were used to identify vascular plants specimens. The usefulness of DNA barcoding data in enhancing the performance of a digital identification key was tested on three independent simulated scenarios.In this paper, we investigated how the DNA barcoding can support the modern digital approaches to the identification of organisms, using as a case study a local flora, that of Mt. Valerio, a small hill near the centre of Trieste . The core barcode markers . The  In biology, the identification process consists of assigning an existing taxon name to a specimen. Although related to classification, which is the job of taxonomists, identification belongs to a different operational process During the last decades, several molecular approaches to the identification of organisms have been explored cox1 (cytochrome c oxidase subunit 1) as a standard region. However, as far as plants are concerned, there was no strong consensus on which DNA regions should be used . The Plant Working Group of the Consortium for the Barcode of Life (http://www.barcoding.si.edu/plant_working_group.html) suggested the use of two plastidial coding regions, the rbcL and matK, as core-barcode for plant identification trnH-psbA, could be used for the analysis of closely related taxa DNA barcoding is based on three keystones of modern taxonomy: molecularization . This flora is strongly heterogeneous, since it includes one or a few species only for each genus. The DNA barcoding approach is compared to the use of a digital identification key based upon morphological features. The added value of DNA barcoding data to the identification keys produced by FRIDA is discussed, detailing the idea of an innovative integrated identification system, obtained by joining morphological and molecular data.Many DNA barcoding studies on plants analysed the discriminating power of molecular data within relatively homogeneous groups, such as families or genera Hieracium racemosum for rbcL, Koeleria lobata for matK, and Cistus salvifolius, Hieracium racemosum, and Stellaria media for psbA-trnH. The rbcL sequences showed an averaging complete intraspecific identity for all but six species with a maximum of 0.7% for Inula hirta. The matK and trnH-psbA showed a certain degree of intraspecific variability, but K2P values were consistently lower than 2%, as was expected in the case of a strongly heterogeneous local flora. Based on the reduced intraspecific variability for the three markers, and since this work was not meant to generate alpha-taxonomy, we hereafter conducted our analyses on one sample for each species. This strategy was used to define a local DNA barcoding library to evaluate the discriminating power of the tested markers in the plant identification processes.A group of 50 randomly selected taxa was used to evaluate the intraspecific genetic variability for the three markers. The results of this preliminary survey are reported in Inula spiraeifolia, Genista germanica, Trifolium arvense subsp. arvense, and Calamintha nepeta. For the latter, electrophoretic analysis showed partially degraded DNA in the 100–1000 bp range and low yield of DNA extraction (data not shown). As a consequence, marker-specific DNA barcoding libraries were defined on a total of 343 taxa. The rbcL was successfully amplified and sequenced in ca. 98% of the tested samples, and trnH-psbA and matK in ca. 94% of the samples. However, the latter required three different sets of primers . The amplification and sequencing of all three markers was obtained for 300 samples  was obtained from all 347 samples, but 4 species:  primers . AccessimatK, with a mean K2P value of 27.9%  computed on 323 samples, which lead to the definition of 313 Molecular Operational Taxonomic Units (MOTUs) (data not shown). In the 8 congeneric groups, K2P values ranged from 0.4% (Gr5) to 5.2% (Gr2). Complete sequence identity was observed for some taxa of Gr5 , Gr6 (Senecio vulgaris and S. inaequidens) and Gr7 .K2P molecular distance (converted into percentage), was used to evaluate the discriminating power of the three DNA barcoding markers in the total flora and in 8 congeneric groups (G1–8). For each group, morphological characters necessary to achieve a correct identification by using the digital key were also reported . BetweenrbcL sequence showed the lowest genetic variability, with 308 MOTUs on 337 amplified sequences. The average genetic distance on total flora was 10.8%. Complete sequence identity was observed in Gr1 (Acer campestre and A. pseudoplatanus), Gr2 (Euphorbia maculata and E. nicaeensis subsp. nicaeensis), Gr3 (Geranium sanguineum and G. molle), Gr4 , Gr6 (Senecio vulgaris and S. inaequidens), and Gr7 . K2P distances in Gr5 (Prunus samples) were lower than 1%, and five accessions were indistinguishable  to 20.6% (Gr2). Few taxa in Gr5 , Gr6 (Senecio vulgaris and S. inaequidens) and Gr7  had identical trnH-psbA sequences.The amplification of rbcL alone  can reduce the uncertainty in the identification process, leading to the identification of a minimum of 92.7% (Scenario C), up to 95.0% (Scenario B) of the species. matK identifies from 95.0% (Scenario C) to 98.0% (Scenario B) of the species, while trnH-psbA has the highest values of discrimination (up to 100%). For this marker, MOTUs always correspond to the related taxa in two out of three scenarios.On the basis of the three simulated scenarios, the digital key returned three groups of 37, 105 and 41 species respectively, which are undistinguishable due to missing characters in the specimens see . Table 3trnH-psbA did not improve the discriminating success with respect to the use of the last marker alone , the matK amplification system requires some improvements , in order to be effective when applied as a universal DNA barcode region for plants. On the other hand, the rbcL marker, which is easy to amplify, sequence and align, has a limited discrimination power, especially when among closely related species. These results are in agreement with the Fourth International Barcode of Life Conference (www.dnabarcodes2011.org/), during which matK and rbcL coding regions were, in any case, confirmed as universal core-barcodes.However, there exist some constraints to the use of the two core-barcode markers. The trnH-psbA region as an additional marker, especially when DNA barcoding is applied to closely related plant taxa. This region has highly conserved PCR priming sites, and a non-coding region with high numbers of substitutions. Hence, trnH-psbA can be a suitable marker to discriminate among closely related species. Although previous research reported the frequent occurrence of stutter PCR products for trnH-psbA due to mononucleotide repeats As stated by other investigators trnH-psbA) could be useful to decide whether to incorporate them into core-barcode when dealing with plants.In addition, as pointed out by A practical result of our investigation concerns the sampling strategy adopted to develop a DNA barcoding database useful for plant identification. In the local context, the low genetic intraspecific variability suggests that a DNA barcoding profile for only one individual per species is enough “to assist in the process of identifying unknown specimens to known species” Some authors suggested a superiority of molecular tools in comparison with “classic” identification keys based on morphological data Morphology can be useful to discriminate among closely related taxa, as shown in Prunus spinosa subsp. spinosa and P. domestica subsp. insititia) were readily distinguished by morphological features, while species belonging to the genera Mentha (i.e. M. longifolia and M. pulegium subsp. pulegium), and Inula (i.e. I. hirta and I. spiraeifolia) showed similar morphological features, but were successfully distinguished by DNA barcoding. The integration of the two approaches, with the development of a multi-parametric identification system, may enhance the overall effectiveness, and represent a real advancement in plant identification. Such a system could be used in very different scenarios, from the identification of plant parts While identification keys based on morphology discriminate among morphospecies, DNA barcoding discriminates among MOTUs, and sometimes these two “entities” could not match http://www.keytonature.eu/wiki/).Software such as FRIDA has the potential to integrate different data sources, including the capacity of using ‘filters’ to automatically reduce a large key by retaining only subsets of species which share a certain character. The inclusion of molecular characters as ‘filters’ does not present any technical problem. In an integrated system, DNA barcoding data could be used as a ‘filter’ by the software which produces and manages the interactive morphological keys. When molecular data are not sufficient to identify a plant at species level, the system will automatically produce a morpho-anatomical key only to the species which are not distinguished by barcode data. This would be an innovative approach to digital identification, which combines morphological and molecular data, overcoming the limits of both approaches, with the potential of becoming the core of a standardised protocol useful in biodiversity surveys, as a new integrated plant identification system, as already proposed by matK, rbcL and trnH-psbA), the extent of their intraspecific variability, and their discriminating power on a subset of congenerics were investigated. In the second step, the performance of each marker (or of different combinations of them) was quantitatively estimated in three simulated scenarios in which the digital key could fail.The area of Mt. Valerio , which hosts a well-known flora, was selected to create a digital identification key and a DNA barcoding library. To investigate how molecular-based data could improve the effectiveness of a digital key, a two-step pipeline was followed. In the first step, the universality of three DNA barcode markers  in the north-east suburban area of Trieste. It is characterized by a submediterrean climate, strongly influenced by the Adriatic Sea, with a dry summer, rainy autumn and spring, and occasional frosts in winter. Average annual precipitation is 1016,9 mm, and average annual temperature is 14,1°C. Prevailing winds are the cold and dry Bora, blowing in winter from east-north-east, and the southern, mild and humid Scirocco. The substratum is Flysch, a base-rich formation of sandstones and marls. The whole survey area has a surface of ca. 0,25 kmhttp://dbiodbs.units.it/carso/chiavi_pub21?sc=77, in Italian and English. It can be used with two query interfaces The digital key to the flora of Mt. Valerio was generated by FRIDA, on the basis of morpho-anatomical, ecological and distributional data, plus original images and drawings, deriving from a floristic list by Poldini et al. (unpublished data). The key is freely accessible online at the address www.biorepositories.org), and the data standards for BARCODE Records in INSDC . All experiments, procedures and ethical issues were conformed to the competent national ethical bodies. No specific permits were required for sampling activities, which were conducted in a non protected area, in accordance with the national and regional laws. The location was not privately owned or in any way protected and field studies did not involve endangered or protected species. Specimens and voucher codes are listed in A total of 347 species were sampled in the period 2009–2010. For each individual, young leaves or buds were collected from at least three different individuals, and stored at −20°C. All samples were vouchered as ‘MIB:ZPL’ following the protocol specified by the biorepositories initiative , to obtain high-quality DNA, free of polysaccharides or other metabolites that might interfere with DNA amplification rbcL and matK coding regions and the non-coding trnH-psbA intergenic spacer of plastidial DNA. PCR amplification was performed by using puReTaq Ready-To-Go PCR beads  in a 25 µL reaction according to the manufacturer's instructions. PCR cycles consisted of an initial denaturation step for 7 min at 94°C, 35 cycles of denaturation (45 s at 94°C), annealing , levels of genetic variation were analyzed by using MEGA 4.0 For each group and for each tested marker, DNA barcode sequences were aligned using MUSCLE – default options To simulate situations in which a digital identification key based on morphological features could fail in achieving correct species identification, three different scenarios were defined. A description is given for each scenario, together with the list of characters used in the digital identification key (multi-entry query interface). In all cases, the identification, which should continue with the dichotomous query interface, fails because of missing features on the specimen. The simulations use a special version of the digital key containing only those taxa for which at least a sequence was successfully amplified.Scenario A – The simulation takes into account a specimen of a non laticiferous, terrestrial herbaceous plant with opposite, non-whorled, entire leaves, collected out of its flowering period. Characters used in the multi-entry query interface of the digital key are: 1) herbaceous plant or a small shrub (<50 cm), 2) green, with chlorophyll, 3) without spines, 4) terrestrial, 5) leaves opposite, 6) leaves simple, 7) leaves not whorled.The result from the multi-entry query interface is a list of 37 taxa, the identification of which can continue by using the dichotomous query interface, which asks whether the plant is laticiferous or not (answer: no) and then whether flowers have petals or not. The identification process, at this point, cannot be continued, because the specimen does not have flowers.Scenario B - The simulation takes into account a specimen of a non laticiferous, terrestrial herbaceous plant with alternate, simple leaves, collected out of its flowering period. Characters used in the multi-entry query interface of the digital key are: 1) herbaceous plant or a small shrub (<50 cm), 2) green, with chlorophyll, 3) without spines, 4) terrestrial, 5) with leaves, 6) leaves not opposite, 7) leaves entire, 8) leaves not whorledThe result from the multi-entry query interface is a list of 105 taxa. The dichotomous query interface asks whether the flowers have petals or not. The identification process, at this point, cannot be continued, because the specimen does not have flowers.Scenario C - The simulation takes into account a specimen of a tree collected in late Autumn, with dry fruits but missing leaves. Characters used in the multi-entry query interface are: 1) tree, woody climber or shrub >50 cm tall, 2) not a woody climber, 3) deciduous, 4) terrestrial, 5) fruit dry.The result from the multi-entry query interface is a list of 41 taxa. The identification continues with the dichotomous interface, which asks whether the leaves are opposite or not. The identification process, at this point, cannot be continued, because the specimen does not have leaves.Table S1List of the analysed plants collected from Mt. Valerio flora. For each sample the voucher number (V.N.), the species name (Nomenclature follows (DOC)Click here for additional data file.Table S2Evaluation of intraspecific genetic variability. For a subset of 50 plant species from the Mt. Valerio the mean values of intraspecific variability and the standard error for the three tested makers are provided. Sampling details can be retrieved from (DOCX)Click here for additional data file.


The Second China National Sample Survey on Disability in 2006 showed that the participation rate of disabled Chinese rural individuals in social medical insurance participation was less than 30%. However, there has been limited number of studies on the influencing factors, especially contextual factors, affecting their participation in social medical insurance. Therefore, this study aimed to analyze the factors influencing the participation of disabled rural individuals in social medical insurance, including contextual factors.Based on data derived from the Second China National Sample Survey on Disability, chi-square test and two-level logistic regression model were used to analyze the influencing factors.The results showed that the disabled rural individuals in the New Rural Cooperative Medical System pilot counties who lived in communities with rehabilitation stations or with higher per capita income of villagers were more likely to participate in the social medical insurance. Meanwhile, those employed, with less severe disability degree or with less severe barriers in participation in society were more likely to participate in the social medical insurance.Contextual factors including economic and policy contexts were important factors influencing their participation in social medical insurance before 2006 in China. Unemployment, severer disability degree and social isolation might also prevent them from gaining equal access to social medical insurance. Some international organizations and countries have introduced programs or laws to protect their rights. World Program of Action Concerning Disabled Persons[Disability Persons approved Persons passed i Persons enacted  Persons emphasizThere are 650 million disabled individuals all over the world, accounting for 10% of the world’s total population and the Disabilities may cause diseases that might lead to poverty and poverty might in return cause diseases that reinforce disabilities. Therefore, a vicious circle shadows the life of some disabled individuals. In factStudies on social medical insurance of the disabled are quite few in China, especially before 2006 when the lack of survey data made it impossible to carry out comprehensive studies on social medical insurance of disabled individuals. Based on the data of the SCNSSD, some domestic scholars carried out studies on medical security-18. HoweThis study was based on the data derived from SCNSSD. In order to understand the latest situation of the disabled individuals so as to help formulate development plans and relevant regulations, China carried out the SCNSSD in 2006. There are many definitions for “disabled person/person with disability”. The SCNSSD and this study adopted the concept of disabled persons in Law of the People’s Republic of China on the Protection of Disabled Persons. Based on these, the disabled person refers to people who have lost all or part of the ability to engage in certain activities in the normal way as a result of certain tissue or function loss or psychological, physiological or anatomical abnormality. This definition also includes people with visual, hearing, speech, physical and mental disabilities along with mental retardation or multiple disabilities.The SCNSSD was a population-based, nationally representative survey that was conducted in 2006 in China. The target population was households that were sampled from 31 provinces, autonomous regions and municipalities in China .In order to get the actual number of households and population, the investigators entered into the survey communities ahead of time. The “Survey Reference Book” was formulated based on the actual situation. On-site registration consisted of household survey and disability assessment. As for the household survey, the households were investigated under the guidance of “Survey Reference Book” using “Survey Questionnaire for Household”. As for the disability assessment, doctors of various specialties combined the stationary and household surveys to assess the disability on an individual basis. Finally, the completed survey questionnaires were reviewed comprehensively by investigators and doctors.The comparison of the age structure, sex ratio and other demographic indicators of the SCNSSD data in 2006 with the data from the 1% Population Sample Survey in 2005 showed that the sample had a good representativeness.The post-survey quality check showed that the omission rate was 1.31% for the registered population and 1.12% for the disabled population. This was an indication of a high integrity.Under the 95% and 90% confidence levels, the allowable errors for the proportion of surveyed disabled population to the national population were 0.97‰ and 0.80‰ respectively. According to some experimental data provided by the United Nation (UN) experts, the data with a relative error of less than 10% was considered reliable. Based on this, the SCNSSD data was reliable.The Whipple’s index was 96.00 for the males and 96.10 for the females, which were very close to 100. Furthermore, the Myer’s index was 1.55, which was less than 5, indicating that the SCNSSD data was accurate.In summary, the SCNSSD data had a high quality.The dependent variable was Participation in Social Medical Insurance, which was binary .The individual-level variables included demographic indicators , economic status indicator (Per Capita Household Income (PCHI)) and disability status indicators .Gender was a dummy variable, coded as 1 and 2, representing male and female, respectively. Age was a continuous variable, representing participant’s age at the time of the baseline interview. Educational level was a dummy variable, coded as 1, 2 and 3, representing college or above, primary, high and technical secondary school and primary school and below, respectively. Marital status was a dummy variable, coded as 1 and 2, representing in marriage and not in marriage, respectively.Employment was a dummy variable, coded as 1 and 2, representing employed and unemployed, respectively.PCHI was based on the information gathered from the 1% of the census (2005), which indicated a poverty level of 683 Yuan. The aveDisability degree was a dummy variable, coded as 1, 2, 3 and 4, representing grade-1 (most severe), grade-2, grade-3 and grade-4 (least severe), respectively. Participation in society was a dummy variable, coded as 1, 2 and 3, representing none/mild barriers, moderate barriers and severe barriers, respectively.Community-level variables were indicators of the communities where the disabled lived, including an economic status indicator (Per Capita Income of the Villagers in 2005 (PCIV)), a policy indicator ) and other contextual indicators  and rehabilitation stations).The average PCIV in our sample was 2,800 Yuan. Therefore, in our analysis we used three income levels, coded as 1, 2 and 3, representing 683 or less, 684–2,800 and 2,801 or more, respectively. NPC was a dummy variable, coded as 1 and 2, representing NPC and non-NPC, respectively. SCD was a dummy variable, coded as 1 and 2, representing with SCD and without SCD, respectively. Rehabilitation station was a dummy variable, coded as 1 and 2, representing with rehabilitation stations and without rehabilitation stations, respectively.More than 2,300 Yuan, grade-4, severe barriers, more than 2,801 Yuan, non-NPC, without SCD and without rehabilitation station served as the reference.x2-test was used to examine the independent associations between rural disabled persons’ social medical insurance participation rate and each independent variable.The methods of calculating frequencies and averages were used to describe the independent variables. The Because of the hierarchical structure of the data and the discrete outcome, multilevel logistic regression analysis, a type of the Generalized Linear Mixed Model, was useσ2μ0 = − 2.72, p < 0.0001). An ICC of 0.8264 was very large and indicated that 82.64% of the total variation in the mean outcome measure was caused by variation between the survey sites. The combined model waspij was the probability of rural disabled persons’ participation in social medical insurance and uj0  was the random variation in the level-1 intercept across groups (survey sites in this study), which represents the deviations of the jth group’s mean logit from the overall mean logit.In step-1, a null model was developed to assess the within-group homogeneity. The results of running an empty model showed a significant between-group variation in the mean outcome measure  and 57,189 were female (50%). As such, the other individual-level and community-level characteristics of the participants are shown in Table Among the rural disabled persons investigated in 2006, only 32,374 participated in social medical insurance, accounting for 28%. Compared with the disabled rural individuals who were unemployed, with lower PCHI, with more severe disability degree or with more severe barriers in participation in society respectively, those employed with higher PCHI, with less severe disability degree or with less severe barriers in participation in society had significantly higher social medical insurance participation rates p < 0.001). As for the community-level variables, the rural disabled persons in the area with rehabilitation stations, with SCD, in NPC or with a higher PCIV had significantly higher social medical insurance participation rates than those in the area without rehabilitation stations, without SCD, in non-NPC or with lower PCIV . The community-level variables were added to model 1. The results showed that the odds of participating in social medical insurance of rural disabled persons in the area with RS, in NPC and with higher PCIV were higher, with statistical significance. However, SCD did not influence rural disabled persons’ participation in social medical insurance. The individual-level variables including disability degree, participation in society, PCHI and employment were added to model 2. All the results were statistically significant with a −2*Log Likelihood (LL) of 658,390.6. Therefore, the odds of participating in social medical insurance of rural disabled persons with more severe disability degree, more severe barriers in participation in society, lower PCHI or no job were lower.The individual-level background variables such as gender and age were added to model 3 as covariates. The results showed that the age was not statistically significant, thus it was removed from the analysis. Finally, the -2LL of model 3 was 658,366.3. The difference in -2LL (likelihood ratio (LR) = 24.3, df = 2, p < 0.0001) between model 3 and 2 indicated that the fit of model 3 improved over model 2 with an acceptable statistical significance. In order to discuss how NRCMS pilot policy mediated the effects of disability degree on rural disabled persons’ social medical insurance participation, the cross-level interaction between NPC and disability degree was added to model 4. The results showed that the interaction was not statistically significant with a -2LL of 658,387.4 for model 4. The difference in -2LL  between model 4 and 3 indicated that the fit of model 3 was better. Meanwhile, the cross-level interaction between disability degree, PCHI, participation in society, employment and other community-level variables were also tested but showed no statistical significance. Therefore, model 3 was chosen in the end.Based on the results gathered from the assessment of model 3, the contextual factors including policy and economic contexts had important influence on rural disabled persons’ participation in social medical insurance in China. A statistically significant relationship was found in 3 community-level variables. More specifically, rural disabled persons in NPC had higher odds of participating in social medical insurance as compared to those in non-NPC . Furthermore, rural disabled persons in the area with rehabilitation stations had higher odds of participating in social medical insurance than those without rehabilitation stations . Rural disabled persons with higher PCIV were more likely to participate in social medical insurance. The rural disabled persons with PCIV of less than 683 Yuan or even l,684 to 2,800 Yuan had lower odds of participation in social medical insurance than those with more than 2,801 Yuan . As for the disability degree, rural disabled persons with more severe disability were less likely to participate in social medical insurance. Rural disabled persons with a grade-1, 2 or 3 disability had lower odds of participating in social medical insurance as compared to those with a grade-4 disability . Rural disabled persons with no barrier, mild or moderate barriers in participation in society had higher odds of participating in social medical insurance as compared to those with severe barriers . In 2010, China Disabled Persons’ Federation granted us an amount of funding, which was managed by Peking University Health Science Center. Since the funding was limited, it could not cover the costs of this manuscript. Therefore, the authors had to pay a proportion of the cost. The authors declared that they had no other financial competing interests.This paper took the jointed efforts of the three authors. JH conceived of the original idea and participated in its data collection, statistical analysis and writing. XLP participated in the study design, critical revision and coordination. AL participated in the statistical analysis, writing, revision and translation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/13/58/prepub


Raja pulchra, is an economically valuable fish. However, due to a severe population decline, it is listed as a vulnerable species by the International Union for Conservation of Nature. To analyze its genetic structure and diversity, microsatellite markers were developed using 454 pyrosequencing. A total of 17,033 reads containing dinucleotide microsatellite repeat units  were identified from 453,549 reads. Among 32 loci containing more than nine repeat units, 20 primer sets (62%) produced strong PCR products, of which 14 were polymorphic. In an analysis of 60 individuals from two R. pulchra populations, the number of alleles per locus ranged from 1–10, and the mean allelic richness was 4.7. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The Hardy–Weinberg equilibrium test showed significant deviation in two of the 28 single-loci after sequential Bonferroni’s correction. Using 11 primer sets, cross-species amplification was demonstrated in nine related species from four families within two classes. Among the 11 loci amplified from three other Rajidae family species; three loci were polymorphic. A monomorphic locus was amplified in all three Rajidae family species and the Dasyatidae family. Two Rajidae polymorphic loci amplified monomorphic target DNAs in four species belonging to the Carcharhiniformes class, and another was polymorphic in two Carcharhiniformes species.The mottled skate,  Rajidae, 11 skate species belonging to four genera have been identified in Korea to date. Skates are commercially important in Korea, and the mottled skate, Raja pulchra, is the most favored and valuable species because of its superior meat quality [Many sharks and skates are particularly vulnerable to overfishing because of their large size, slow growth, late maturity and low fecundity. Among the 227 species in 25 genera of the family Raja pulchra, a low-boreal subtropical species abundant in the coastal waters of some areas of Korea and Japan, is usually caught at a depth of about 5–100 m [ 5–100 m –4. This  5–100 m .Catch data from Korea shows a 90% decline over a ten-year period. The average annual catch from 1991–1993 was 2700 metric tons, whereas the catch in 2011–2003 was 220 metric tons . As a reMicrosatellite (MS) or simple sequence repeat (SSR) markers are versatile molecular tools for determining parentage, inferring genetic structure and gene flow patterns and for assessing the origins of introduced populations ,10. TradNext generation sequencing (NGS) technologies have revolutionized many life science fields because of its rapid cost-efficient parallel processing of millions of templates . NGS is Based on these advantages, this new technology is expected to replace conventional microsatellite isolation protocols , and theR. pulchra genome using NGS combined with the bioinformatics approach described above. This work will be useful for future resource management of this economically and ecologically important, but vulnerable, species.In this work, we carried out a global analysis of the repetitive fraction of the NGS produced 453,549 reads with an average length of 487 bp; 4,305 contiguous sequences (contigs) were constructed by Newbler  from these reads. The total number of bases was 2,024,317, and the largest contig was 16,906 bp. In addition, we found 307,931 unassembled singleton sequences for a total read of 150,038,230 bp. The total number of reads after trimming of low quality sequence was 288,854, and the sum of the total reads was 148,321,261 bp in length, which was used in a search for SSRs. The number of reads with dinucleotide repeat were: 4164 AT, 6544 CA, 6239 CT, and 86 GC repeats. The sequence and number of 2625 trinucleotide repeats are shown in Thirty two loci containing dinucleotide microsatellites with over nine repeats were selected for further evaluation. Primers were designed for these loci and tested in four individuals. Twenty primer sets (62%) produced strongly amplified PCR products, and 14 showed clear amplification with polymorphic patterns. However, no clear genotype pattern was obtained for five loci, and Rp19-nfrdi was monomorphic in all of the tested individuals. The primer sequences, repeat motifs, annealing temperatures, PCR ranges, number of alleles, and GenBank accession numbers for the 15 new microsatellite loci are summarized in p > 0.05), indicating that the markers were independent. The Hardy–Weinberg equilibrium (HWE) test, indicating the deviation from expected heterozygosity, showed significant deviation in two (Rp3-nfrdi and Rp43-nfrdi) of the 28 single-loci in the Heuksando population after sequential Bonferroni’s correction. Null alleles were presumed in four  of 14 loci. No genetic differentiation between DC and HS populations was detected by FST (=0.013) using all 14 microsatellite markers.Okamejei boesemani, O. kenojei and O. acutispina that belong to the same family, Rajidae. Among these loci, Rp11, Rp16 and Rp53 were polymorphic in all three species. The locus Rp16, which was monomorphic in R. pulchra, was also monomorphic in all three species. Additionally, the monomorphic locus Rp16 was amplified in two fish species, Dasyatis akajei and Uroplophus aurantiacus, belonging to the Dasyatidae family. In contrast, no amplification of other loci was observed in either or both of these species. The Rp3 and Rp16 loci that were polymorphic in the Rajidae family amplified monomorphic target DNAs in four fish species belonging to the class Carcharhiniformes, and the Rp35 locus was polymorphic in two species belonging to this class  from a total of 312,290 contigs contained dinucleotide SSRs, and 33 loci containing a minimum of nine repeat units were used for primer design, which is only 0.2% of the identified SSRs. In the analysis of MS markers reported in plant, only 1% of polymorphic loci identified using NGS were selected by stringent criteria for primer design, compared with 20% of SSRs discovered using a traditional cloning and sequencing process were selected for primer design .To select optimal primers from a large number of candidates, high-quality standards can be applied in loci selection, which can increase the success rate and reduce the time, labor and cost of marker development. For instance, to minimize the risk of null allele amplification, 454 sequence alignments can be used to design primers using loci with a reasonably high depth of coverage at both primer-annealing sites, and that show no evidence of SNPs within the regions . In our Ho = 0.57, He = 0.61; mean allelic number = 4.7) in this study was slightly higher than that of the common skate, Dipturus batis  [FST may suggest that the two populations can be regarded as one population. However, differentiation of genetically related skate population by distance has been observed in northeast Atlantic continental shelf [After sequential Bonferroni correction for multiple tests, significant deviations from HWE in the direction of heterozygote deficiency were detected at only two (Rp3-nfrdi and Rp43-nfrdi) of the 14 tested loci in the Heuksan-do population. Generally, heterozygote deficiencies increase due to factors such as inbreeding, substructuring of the population sample, or the presence of null alleles. Indeed, our Microchecker analysis revealed the presence of null alleles at four loci, including Rp3-nfrdi and Rp43-nfrdi. The level of genetic diversity (mean r = 3.5) . No geneal shelf . To redual shelf ,34. Moreal shelf .R. pulchra, four (40%) were polymorphic in other species within same family, which is comparable to previous data [Dasyatididae, produced monomorphic or polymorphic PCR products in Carcharhiniformes. Although the numbers of genera and samples for each genus are limited, these results suggest the possibility that cross-species polymorphic markers can be developed in fish species using the pyrosequencing technique with large number of SSR candidates for marker development.With large numbers of SSR candidates identified by pyrosequencing, the development of cross-species markers has been attempted in diverse organisms . The marous data . HoweverR. pulchra around the Korean Peninsula, especially in the Yellow Sea, is due to many factors such as overfishing and destruction of their natural habitat. However, there have been no conservation measures in place for this species. Therefore, the molecular markers developed in this study will be useful for future assessment and monitoring of population trends of this species. In addition, this study suggests that pyrosequencing methods will be useful for SSR development in aquatic organisms, and may be preferable to the traditional labor- and time-intensive cloning methods.The rapid population decline of R. pulchra samples were collected around the Korean islands: 30 from the Daecheong-do population and 30 from the Heuksan-do population. Muscle tissue samples were preserved in 100% ethanol at the sampling site and then transported to the laboratory for DNA extraction. Total DNA was isolated from each sample using a MagExtractor MFX-6100 automated DNA extraction system . The extracted genomic DNA was quantified using a Nanodrop ND-1000 spectrophotometer  and stored at −20 °C until genomic DNA pyrosequencing. For the cross-species transferability test, DNA was extracted by the same method from ethanol-fixed tissues of nine related species belonging to four families within two classes that had been stored at the National Fisheries Research and Development Institute, Busan.A total of 60 wild DNA sequencing was performed with 454 pyrosequencing on a Genome Sequencer FLX-454 System (GS FLX sequencer). Sample preparation and DNA sequencing was performed according to the manufacturer’s instructions . DNA sample was prepared from one individual and sequencing was conducted using a half of a 454 plate at the National Instrumentation Center for Environmental Management (NICEM) of Seoul National University.The raw reads from the GS FLX 454 were assembled using the Newbler 2.6 software and the assembled contigs consensus and unassembled singleton sequences were merged to discover SSRs on the genome sequence. The sequence was filtered with high quality score using the Less Useful Chunks Yank (LUCY) 1.20p. A modified “SSR_finder.pl” perl program was then used to detect di- and tri-SSR markers. A pair of primers flanking each SSR was designed using Primer3 software . The priEx taq DNA polymerase , 1× PCR buffer, 0.2 mM dNTP mix, 10 pmol each primer , and 100 ng template DNA, using a PTC 200 DNA Engine . PCR conditions were as follows: 11 min at 95 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at the annealing temperature listed in Newly-designed PCR primer pairs were tested to optimize annealing temperatures; a gradient PCR with a 50–60 °C annealing temperature range was performed on a set of samples from eight individuals. PCR amplification was performed in a 10 μL reaction mixture containing 0.25 U p-values for both analyses were obtained using a sequential Bonferroni test for multiple comparisons. MICROCHECKER version 2.2.3 [AR) as a standardized measure of the number of alleles per locus, independent of the sample size, was calculated using FSTAT version 2.9.3 [FST estimates and genetic distances between each pair of populations.The number of alleles per locus, allele frequency, and heterozygosity were calculated using Arlequin version 3.0 . Tests fon 2.2.3  was usedon 2.9.3 . A possiR. pulchra, which is listed as a “vulnerable” species by the IUCN. A comparably high primer-to-marker conversion ratio (62%) was achieved by this method. In the cross-species amplification, over 90% of the markers were amplified in the fishes belonging to the same family but the success ratio was much less in the different family. The molecular markers developed in this study can be used for future management of this economically and ecologically important species.The pyrosequencing method was applied in the development of MS markers for


Chylothorax is a rare clinical entity characterized by a milky white aspirate with increased triglyceride levels. The commonest etiology is malignancy and trauma, and bilateral chylothorax, secondary to tuberculosis, is an extremely rare cause, as observed in the present case. Chylothorax is a relatively rare cause of a pleural effusion and it occurs when chyle, characterized by high-triglyceride and low-cholesterol concentrations, is found in the pleural space and is usually associated with neoplasms or trauma to the thoracic duct.2 The devA 15-year-old female was admitted to our department with the complaints of breathlessness, and off and on fever for 1 month. She came from a nonendemic zone of filariasis in Uttar Pradesh. The resting pulse rate was 92/min and blood pressure was 112/74 mmHg. There was no history of contact with a tuberculosis patient. Her physical examination revealed hard, mobile lymph nodes of size 2 × 2 cm approximately, present bilaterally in the cervical region, and pallor. On chest examination, there was stony dull note localized to the bilateral infrascapular, lower axilla. Rest of the systems were within normal limits.3; differential leukocyte count was neutrophils 20, lymphocytes 80, pleural fluid triglyceride 535 mg%, and pleural fluid cholesterol 24.8 mg%; pleural fluid of the left side revealed protein 4.0 g%, sugar 40 mg%, and total leukocyte count 3280 cells/mm3; differential leukocyte count was neutrophils 34, lymphocytes 66, pleural fluid triglyceride 188.0 mg%, and pleural fluid cholesterol 24.0 mg% 37]Primary treatment in the case of chylothorax should be directed toward the correction of malnutrition and compromised immunologic status which is due to repeated pleural fluid aspirations of chyle with its high levels of protein, fat, electrolytes, and lymphocytes. The defeIn our case, diagnosis of chylothorax was established on typical pleural fluid color, high pleural fluid triglyceride level, high ratio of pleural fluid to serum triglyceride, and low ratio of pleural fluid to serum cholesterol. She responded well to antituberculous treatment.Thus, we should remember that while treating any patient with chylothorax, the probable diagnosis of tuberculosis should be kept in mind.


Disturbances in feedback processing and a dysregulation of the neural circuit in which the cingulate cortex plays a key role have been frequently observed in depression. Since depression is a heterogeneous disease, instead of focusing on the depressive state in general, this study investigated the relations between the two core symptoms of depression, i.e., depressed mood and anhedonia, and the neural correlates of feedback processing using fMRI. The focus was on the different subdivisions of the anterior cingulate cortex (ACC). Undergraduates with varying levels of depressed mood and anhedonia performed a time-estimation task in which they received positive and negative feedback that was either valid or invalid . The rostral cingulate zone (RCZ), corresponding to the dorsal part of the ACC, was less active in response to feedback in more anhedonic individuals, after correcting for the influence of depressed mood, whereas the subgenual ACC was more active in these individuals. Task performance was not affected by anhedonia, however. No statistically significant effects were found for depressed mood above and beyond the effects of anhedonia. This study therefore implies that increasing levels of anhedonia involve changes in the neural circuitry underlying feedback processing. Major depressive disorder (MDD) is a serious mental illness, characterized by at least one of two core symptoms: depressed mood and anhedonia . MDD affects both affective and cognitive functioning. One of the deficits in MDD in which cognition and affect both play a role is impaired feedback processing. Behavioral studies have shown that depressed individuals are hypersensitive to negative feedback. When they make an error or receive negative feedback on their performance, their subsequent performance deteriorates , which can be further subdivided into an anterior (aMCC) and posterior (pMCC) part, and a ventral part (ACC), which can be further subdivided into a pregenual (pgACC) and subgenual (sgACC) part Vogt, . The aMChypoactive, while brain regions primarily involved in emotion processing, such as the amygdala and sgACC, have been found hyperactive  and validity . In contrast to most tasks, in which the valence and information value of feedback are highly correlated, this paradigm enables us to disentangle emotion processing  from cognitive control . We have reported on this task in previous ERP papers including one that involves clinically depressed individuals  at the time of scanning, and, importantly, resulted in a broad and continuous range of scores on the questionnaires assessing depressed mood and anhedonia, specifically.Respondents were asked to fill out the Dutch translation of the Beck Depression Inventory , substance abuse, use of medication which affects the central nervous system , pregnancy, and any contra-indication for having an MRI-scan. Health criteria were assessed by means of a self-developed questionnaire and contra-indications for MRI were assessed by means of a standard questionnaire from the department of Radiology.M = 23, SD = 3.5), participated in this study.Eventually, 42 healthy volunteers, 26 female, aged between 18 and 32 . Finally, the SHAPS consists of 14 items to be answered on a 1–4 scale, ranging from absolutely agree (1) to absolutely disagree (4) . Higher sum scores indicate higher levels of anhedonia.*”) in the center of a black screen for 2 s. This asterisk was followed by the cue for estimation: a question mark (“?”), which was replaced with another asterisk (1 s) after the estimation. This second asterisk was followed by the feedback stimulus (1 s) , and negative feedback if the response occurred outside the window. Unbeknownst to participants, the window was dynamically adjusted on each trial (±20 ms) to ensure an equal amount of positive and negative feedback stimuli . Valid feedback was based on the participant's performance. Invalid feedback was determined randomly by the computer, with a maximum of three invalid feedback trials in a row. Participants received invalid feedback in 50% of the trials. The emotional expression of the face informed participants that their estimate was correct or incorrect . Finally, in case of incorrect estimates, the gender of the face indicated whether the estimate was too short  or too long . The faces used in this study were from the Ekman and Friesen pictures set , participants were scanned. They were asked to abstain from coffee and tobacco for at least 2 h before scanning. Participants first had to fill out the POMS, and were again given task instructions before entering the scanner. When participants were inside the scanner, the visual stimuli were projected on a screen at the end of the scanner bed, which could be viewed by the participant through a small mirror mounted on the head coil. During the time-estimation task participants responded by pressing the button of a response device with their right index finger. Inside the scanner participants performed several practice trials , after which the first session started, consisting of 120 trials (10 min). After a short break, a second session of the task started which again lasted 10 min. Participants performed 240 trials of the time-estimation task inside the scanner. After these two time-estimation sessions a structural scan was obtained, which lasted about 5 min.*-weighted images were acquired in 26 axial slices  with a repetition time (TR) of 2000 ms, echo time (TE) of 30 ms, field of view (FOV) of 220 mm, and voxels of 1.72 × 1.72 × 3.50 mm. The interval between trials was about 5 s. In each session of 120 trials 310 volumes  were obtained. In addition, five dummy scans were made before the task started in order to obtain a steady-state magnetization.Blood oxygen level-dependent (BOLD) fMRI data were acquired on a 3T GE Healthcare  scanner. For the functional scans a single-shot gradient echo echo-planar imaging (EPI) sequence was used. The T2For anatomical reference, a 3D high-resolution inversion recovery fast spoiled gradient recalled echo T1-weighted sequence was used, which covered the whole brain. One hundred and ninety-two slices were acquired with an effective slice thickness of 0.8 mm, FOV of 250 mm, and voxels of 0.49 × 0.49 × 0.80 mm.For pre-processing and processing of the fMRI data SPM5  was used. Preprocessing of the structural data included manual reorienting, unified segmentation using the Montreal Neurological Institute T1 ICBM template for European brains for gray matter, white matter, and CSF, and normalization using the parameters derived from unified segmentation. Preprocessing of the functional data included manual reorienting, slice time correction, realignment using the middle slice as a reference, and unwarping, co-registration , normalization using the parameters derived from unified segmentation, and smoothing using a Gaussian kernel of 8 mm full width at half maximum, and a high-pass filter of 128 s for temporal smoothing.Performance data were analyzed by partial correlation analyses assessing the relation between POMS-depression scores and the percentage of correct adjustments after valid negative feedback and the percentage of “correct” adjustments after invalid negative feedback, while correcting for SHAPS-anhedonia scores, and vice versa .For the fMRI analyses, a model was made in which the preprocessed fMRI data were coupled to the vectors of feedback onset of each condition  in both task sessions. Then two t-contrasts were computed that were used for the whole-brain analyses only: positive—negative feedback , and valid—invalid feedback . The individual contrast images resulting from these contrasts were used in a second-level whole-brain analysis.P < 0.05 corrected with the family-wise error (FWE) approach. The Automated Anatomical Labeling (AAL) atlas  analyses. Four ROI analyses were performed using MarsBaR 0.41  separately. For each ROI, the extracted beta-values of each participant were exported to SPSS, and subsequently analyzed using valence (positive or negative feedback) and feedback-validity  as within-subjects factors in repeated-measures ANOVAs with mean-centered POMS-depression and SHAPS-anhedonia scores simultaneously added as covariates. Effects of lateralization in the RCZ and NAcc are not reported.One student did not perform according to task instructions, and was excluded from all analyses. Therefore, 41 participants were included in the analyses.M = 187, SD = 96), and SHAPS scores ranged from 14 to 34 . The POMS-depression scores and SHAPS-anhedonia scores correlated positively with each other .POMS-depression scores ranged from 8 to 457 , indicating that participants with higher scores of depressed mood performed slightly worse after receiving valid negative feedback. No associations were found with anhedonia.Participants had a mean estimation time of 1053 ± 87 ms, and, as expected, they adjusted their behavior more often in response to valid negative feedback than to invalid negative feedback (85 ± 8% vs. 52 ± 6%). The partial correlation analyses showed a marginal negative correlation between depressed mood and percentage of correct behavioral adjustments after valid negative feedback  = 8.1, P = 0.007, η2p = 0.17; see Figure positive feedback than in response to negative feedback , and was more active in response to positive feedback than in response to negative feedback . In addition, valence and validity interacted in the NAcc ; the effect of valence was strongest in the valid condition  = 3.1, P = 0.084, η2p = 0.08], and with the effect of valence in the NAcc .The purpose of this study was to examine the separate influences of depressed mood and anhedonia on the four predefined ROIs in relation to these task effects. No statistically significant effects were found for depressed mood above and beyond the effects of anhedonia. Depressed mood only marginally interacted with the effect of validity in the RCZ . Higher levels of anhedonia were associated with decreased activity in the RCZ, independent of task condition  = 4.2, P = 0.048, η2p = 0.10; Figure F = 3.7, P = 0.064, η2p = 0.09]. Surprisingly, no relationship was found between anhedonia and activity in the NAcc.Anhedonia, on the other hand, did influence feedback processing. In the RCZ we found a main effect of anhedonia, after correction for variation in depressed mood [n Figure . Also inThe current study investigated the relationship between the two core symptoms of depression, i.e., depressed mood and anhedonia, and behavioral and neural responses to valid and invalid positive and negative feedback. In this student sample, after correcting for the influence of anhedonia, no statistically significant effects were found of depressed mood on feedback processing. Anhedonia, on the other hand, was negatively correlated with activity in the RCZ, and positively correlated with activity in the sgACC in response to feedback stimuli in general, after correcting for the influence of depressed mood  model, was recently developed by Alexander and Brown  processing of feedback-valence is inherent to (aberrant) emotion processing.There are several limitations that need to be addressed. First, as expected, anhedonia and depressed mood were correlated, making it difficult to completely disentangle their unique contributions to the present results. When correcting effects of depressed mood for anhedonia, effects did not reach significance, e.g., the interaction between depressed mood and validity of feedback in the RCZ. Although it is unnatural to select participants on the basis of distinct depressed mood and anhedonia scores, it might be useful in future studies to do so. In addition, larger sample sizes may be effective in disentangling their unique effects. Second, we used emotional faces as feedback stimuli, which may cause some concern about whether found effects are due to feedback processing or to emotional face processing In conclusion, our findings suggest that anhedonia, rather than depressed mood, affects feedback processing at the neural level. Anhedonia was associated with a decreased response of the RCZ to feedback and with an often-reported hyperactive sgACC in depression. Our results imply that increasing levels of anhedonia involve changes in the neural circuitry underlying feedback processing. These atypical neural responses might render subjects vulnerable to depression.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.