Endosomal vesicle fusion machinery is involved with the contractile vacuole in Dictyostelium discoideum.

Contractile vacuoles (CVs), enigmatic osmoregulatory organelles, share common characteristics, such as a requirement for RAB11 and high levels of V-ATPase. These commonalities suggest a conserved evolutionary origin for the CVs with implications for understanding of the last common ancestor of eukaryotes and eukaryotic diversification more broadly. A taxonomically broader sampling of CV-associated machinery is required to address this question further. We used a transcriptomics-based approach to identify CV-associated gene products in Dictyostelium discoideum. This approach was first validated by assessing a set of known CV-associated gene products, which were significantly upregulated following hypo-osmotic exposure. Moreover, endosomal and vacuolar gene products were enriched in the upregulated gene set. An upregulated SNARE protein (NPSNB) was predominantly plasma membrane localised and enriched in the vicinity of CVs, supporting the association with this organelle found in the transcriptomic analysis. We therefore confirm that transcriptomic approaches can identify known and novel players in CV function, in our case emphasizing the role of endosomal vesicle fusion machinery in the D. discoideum CV and facilitating future work to address questions regarding the deep evolution of eukaryotic organelles.

Novel insights in the relationship of gut microbiota and coronary artery diseases.

Atherosclerosis is a chronic, progressive, inflammatory disease in the vasculature and is common in both coronary and peripheral arteries. Human beings harbor a complex and dynamic population of microorganisms defined as the microbiota. Importantly, alterations in the bacterial composition (dysbiosis) and the metabolic compounds produced by these bacteria have been associated with the pathogenesis of many inflammatory diseases and infections. There is also a close relationship between intestinal microbiota and cardiovascular diseases. The aim of this review was to analyze how changes in the gut microbiota and their metabolites might affect coronary artery diseases. The most representative groups of bacteria that make up the intestinal microbiota are altered in coronary artery disease patients, resulting in a decrease in Bacteroidetes and an increase in Firmicutes. In relation to metabolites, trimethylamine-N-oxide plays an important role in atherosclerosis and may act as a cardiovascular risk predictor. In addition, the use of probiotics, prebiotics, diet modulation, and fecal transplantation, which may represent alternative treatments for these diseases, is thoroughly discussed. Finally, the role of lipid-lowering treatments is also analyzed as they may affect and alter the gut microbiota and, conversely, gut microbiota diversity could be associated with resistance or sensitivity to these treatments.

Global longitudinal strain assessment of cardiac function and extravascular lung water formation after diving using semi-closed circuit rebreather.

PURPOSE: The aim of the present investigation is to study the relationship of ventricular global longitudinal strain (GLS) and ultrasound lung comets (ULC) formation to establish a link between extravascular pulmonary water formation and cardiac contractile dysfunction. METHODS: This is a prospective observational study including 14 active military divers. The subjects performed two sea dives of 120 min each with a semi-closed SCUBA circuit at 10 m depth. Divers were examined at baseline, 15 min (D1) and 60 min (D2) after diving. The evaluation included pulmonary and cardiac echography (including speckle tracking techniques). Blood samples were drawn at baseline and after diving, assessing hs-TnT and Endothelin-1. RESULTS: ULC were detected in 9 (64.2%) and 8 (57.1%) of the subjects after D1 and D2 respectively. No differences were found in right and left ventricular GLS after both immersions (RV: Baseline: - 17.9 4.9 vs. D1: - 17.2 6.5 and D2: - 16.7 5.8 s-1; p = 0.757 and p = 0.529; LV: Baseline: - 17.0 2.3 vs. D1: - 17.4 2.1 and D2: - 16.9 2.2 s-1; p = 0.546 and p = 0.783). However, a decrease in atrial longitudinal strain parameters have been detected after diving (RA: Baseline: 35.5 9.2 vs. D1: 30.3 12.8 and D2: 30.7 13.0 s-1; p = 0.088 and p = 0.063; LA: Baseline: 39.0 10.0 vs. D1: 31.6 6.1 and D2: 32.4 10.6 s-1; p = 0.019 and p = 0.054). CONCLUSION: In the present study, no ventricular contractile dysfunction was observed. However, increase pulmonary vasoconstriction markers were present after diving.

Genomics and transcriptomics yields a system-level view of the biology of the pathogen Naegleria fowleri.

BACKGROUND: The opportunistic pathogen Naegleria fowleri establishes infection in the human brain, killing almost invariably within 2 weeks. The amoeba performs piece-meal ingestion, or trogocytosis, of brain material causing direct tissue damage and massive inflammation. The cellular basis distinguishing N. fowleri from other Naegleria species, which are all non-pathogenic, is not known. Yet, with the geographic range of N. fowleri advancing, potentially due to climate change, understanding how this pathogen invades and kills is both important and timely. RESULTS: Here, we report an -omics approach to understanding N. fowleri biology and infection at the system level. We sequenced two new strains of N. fowleri and performed a transcriptomic analysis of low- versus high-pathogenicity N. fowleri cultured in a mouse infection model. Comparative analysis provides an in-depth assessment of encoded protein complement between strains, finding high conservation. Molecular evolutionary analyses of multiple diverse cellular systems demonstrate that the N. fowleri genome encodes a similarly complete cellular repertoire to that found in free-living N. gruberi. From transcriptomics, neither stress responses nor traits conferred from lateral gene transfer are suggested as critical for pathogenicity. By contrast, cellular systems such as proteases, lysosomal machinery, and motility, together with metabolic reprogramming and novel N. fowleri proteins, are all implicated in facilitating pathogenicity within the host. Upregulation in mouse-passaged N. fowleri of genes associated with glutamate metabolism and ammonia transport suggests adaptation to available carbon sources in the central nervous system. CONCLUSIONS: In-depth analysis of Naegleria genomes and transcriptomes provides a model of cellular systems involved in opportunistic pathogenicity, uncovering new angles to understanding the biology of a rare but highly fatal pathogen.

Curvicollide D Isolated from the Fungus Amesia sp. Kills African Trypanosomes by Inhibiting Transcription.

Sleeping sickness or African trypanosomiasis is a serious health concern with an added socio-economic impact in sub-Saharan Africa due to direct infection in both humans and their domestic livestock. There is no vaccine available against African trypanosomes and its treatment relies only on chemotherapy. Although the current drugs are effective, most of them are far from the modern concept of a drug in terms of toxicity, specificity and therapeutic regime. In a search for new molecules with trypanocidal activity, a high throughput screening of 2000 microbial extracts was performed. Fractionation of one of these extracts, belonging to a culture of the fungus Amesia sp., yielded a new member of the curvicollide family that has been designated as curvicollide D. The new compound showed an inhibitory concentration 50 (IC50) 16-fold lower in Trypanosoma brucei than in human cells. Moreover, it induced cell cycle arrest and disruption of the nucleolar structure. Finally, we showed that curvicollide D binds to DNA and inhibits transcription in African trypanosomes, resulting in cell death. These results constitute the first report on the activity and mode of action of a member of the curvicollide family in T. brucei.

Antithrombotic Therapy in Patients with Peripheral Artery Disease: A Focused Review on Oral Anticoagulation.

Peripheral artery disease (PAD) is a major cause of morbidity and mortality but it is usually underdiagnosed and undertreated. Patients with PAD present dysregulated procoagulant, anticoagulant, and fibrinolytic pathways leading to arterial and venous thrombosis. The risk of several ischemic-related complications could be mitigated with appropriate antithrombotic therapy, which plays a central role in all types of PAD. For years, antiplatelets have been indicated in patients with symptomatic PAD or those who have undergone revascularization. Unfortunately, a non-negligible proportion of patients with PAD will suffer from adverse events during the follow-up, even despite proper medical therapies for the prevention of PAD complications. Thus, there is room for improving clinical outcomes in these patients. Given the implication of both, primary and secondary hemostasis in arterial thrombosis and the pathophysiology of PAD, the combination of antiplatelets and anticoagulants has emerged as a potential antithrombotic alternative to antiplatelets alone. In this narrative review article, we have highlighted the most recent evidence about antithrombotic therapy in PAD patients, with a special focus on oral anticoagulation. Certainly, COMPASS and VOYAGER PAD trials have shown promising results. Thus, rivaroxaban in combination with aspirin seem to reduce cardiovascular outcomes with a similar bleeding risk compared to aspirin alone. Nevertheless, results from real-world studies are needed to confirm these observations, and other trials will provide novel evidence about the safety and efficacy of emerging anticoagulant agents.

Effective Tetradentate Compound Complexes against Leishmania spp. that Act on Critical Enzymatic Pathways of These Parasites.

The spectrum and efficacy of available antileishmanial drugs is limited. In the present work we evaluated in vitro the antiproliferative activity of 11 compounds based on tetradentate polyamines compounds against three Leishmania species (L. braziliensis, L. donovani and L. infantum) and the possible mechanism of action. We identified six compounds (3, 5, 6, 7, 8 and 10) effective against all three Leishmania spp both on extracellular and intracellular forms. These six most active leishmanicidal compounds also prevent the infection of host cells. Nevertheless, only compound 7 is targeted against the Leishmania SOD. Meanwhile, on the glucose metabolism the tested compounds have a species-specific effect on Leishmania spp.: L. braziliensis was affected mainly by 10 and 8, L. donovani by 7, and L. infantum by 5 and 3. Finally, the cellular ultrastructure was mainly damaged by 11 in the three Leishmania spp. studied. These identified antileishmania candidates constitute a good alternative treatment and will be further studied.

Simple dialkyl pyrazole-3,5-dicarboxylates show in vitro and in vivo activity against disease-causing trypanosomatids.

The synthesis and antiprotozoal activity of some simple dialkyl pyrazole-3,5-dicarboxylates (compounds 2-6) and their sodium salts (pyrazolates) (compounds 7-9) against Trypanosoma cruzi, Leishmania infantum and Leishmania braziliensis are reported. In most cases the studied compounds showed, especially against the clinically significant amastigote forms, in vitro activities higher than those of the reference drugs (benznidazole for T. cruzi and glucantime for Leishmania spp.); furthermore, the low non-specific cytotoxicities against Vero cells and macrophages shown by these compounds led to good selectivity indexes, which are 8-72 times higher for T. cruzi amastigotes and 15-113 times higher for Leishmania spp. amastigotes than those of the respective reference drugs. The high efficiency of diethyl ester 3 and its sodium salt 8 against the mentioned protozoa was confirmed by further in vitro assays on infection rates and by an additional in vivo study in a murine model of acute and chronic Chagas disease. The inhibitory capacity of compounds 3 and 8 on the essential iron superoxide dismutase of the aforementioned parasites may be related to the observed anti-trypanosomatid activity. The low acute toxicity of compounds 3 and 8 in mice is also reported in this article.

Effective anti-leishmanial activity of minimalist squaramide-based compounds.

In order to evaluate the in vitro leishmanicidal activity of N,N'-Squaramides derivatives, compounds that feature both hydrogen bond donor and acceptor groups and are capable of multiple interactions with complementary sites, against Leishmania infantum, Leishmania braziliensis and Leishmania donovani a series of 18compounds was prepared and assayed on extracellular and intracellular parasite forms. Infectivity and cytotoxicity tests were performed on J774.2 macrophage cells using meglumine antimoniate (Glucantime) as the reference drug. Changes in metabolite excretion by 1H-NMR and the ultrastructural alterations occurring in the parasites treated using transmission electron microscopy (TEM), was analyzed. Compounds 1, 7, 11, 14 and 17 were the more active and less toxic. Infection rates showed that the order of effectiveness was 17 > 11 > 14 > 7 for both L. infantum and L. braziliensis and in the same way, the compound 1 for L. donovani. All these compounds have altered the typical structure of the promastigotes, glycosomes and mitochondria. These severe modifications by the compounds are the ultimate reasons for the alterations observed in the excretion products. The Squaramide 17 (3-(butylamino)-4-((3-(dimetilamino)propyl)(methyl)amino)cyclobut-3-en-1,2-dione) was clearly the most efficient of all compounds. The data appear to confirm that the severe modifications generated in organelles such as glycosomes or mitochondria by the compounds are the ultimate reasons for the alterations observed in the excretion products of all species. The activity, stability, low cost of starting materials, and straightforward synthesis make amino squaramides appropriate molecules for the development of an affordable anti-leishmanial agent.

In vitro leishmanicidal activity of imidazole- or pyrazole-based benzo[g]phthalazine derivatives against Leishmania infantum and Leishmania braziliensis species.

OBJECTIVES: To evaluate the in vitro leishmanicidal activity of imidazole-based (1-4) and pyrazole-based (5-6) benzo[g]phthalazine derivatives against Leishmania infantum and Leishmania braziliensis. METHODS: The in vitro activity of compounds 1-6 was assayed on extracellular promastigote and axenic amastigote forms, and on intracellular amastigote forms of the parasites. Infectivity and cytotoxicity tests were performed on J774.2 macrophage cells using meglumine antimoniate (Glucantime) as the reference drug. The mechanisms of action were analysed by iron superoxide dismutase (Fe-SOD) and copper/zinc superoxide dismutase (CuZn-SOD) inhibition, metabolite excretion and transmission electronic microscopy (TEM). RESULTS: Compounds 1-6 were more active and less toxic than meglumine antimoniate. Data on infection rates and amastigote mean numbers showed that 2, 4 and 6 were more active than 1, 3 and 5 in both L. infantum and L. braziliensis. The inhibitory effect of these compounds on the antioxidant enzyme Fe-SOD of promastigote forms of the parasites was remarkable, whereas inhibition of human CuZn-SOD was negligible. The ultrastructural alterations observed in treated promastigote forms confirmed the greater cell damage caused by the most active compounds 2, 4 and 6. The modifications observed by (1)H-NMR in the nature and amounts of catabolites excreted by the parasites after treatment with 1-6 suggested that the catabolic mechanisms could depend on the structure of the side chains linked to the benzo[g]phthalazine moiety. CONCLUSIONS: All the compounds assayed were active in vitro against the two Leishmania species and were less toxic against mammalian cells than the reference drug, but the monosubstituted compounds were significantly more effective and less toxic than their disubstituted counterparts.

In vitro evaluation of new terpenoid derivatives against Leishmania infantum and Leishmania braziliensis.

The activity of five (1-5) abietane phenol derivatives against Leishmania infantum and Leishmania braziliensis was studied using promastigotes and axenic and intracellular amastigotes. Infectivity and cytotoxicity tests were performed with J774.2 macrophage cells using Glucantime as a reference drug. The mechanisms of action were analysed by performing metabolite excretion and transmission electron microscopy ultrastructural studies. Compounds 1-5 were more active and less toxic than Glucantime. The infection rates and mean number of parasites per cell observed in amastigote experiments showed that derivatives 2, 4 and 5 were the most effective against both L. infantum and L. braziliensis. The ultrastructural changes observed in the treated promastigote forms confirmed that the greatest cell damage was caused by the most active compound (4). Only compound 5 caused changes in the nature and amounts of catabolites excreted by the parasites, as measured by ¹H nuclear magnetic resonance spectroscopy. All of the assayed compounds were active against the two Leishmania species in vitro and were less toxic in mammalian cells than the reference drug.

Leishmanicidal activity of nine novel flavonoids from Delphinium staphisagria.

OBJECTIVES: To evaluate the in vitro leishmanicidal activity of nine flavonoid derivatives from Delphinium staphisagria against L. infantum and L. braziliensis. DESIGN AND METHODS: The in vitro activity of compounds 1-9 was assayed on extracellular promastigote and axenic amastigote forms and on intracellular amastigote forms of the parasites. Infectivity and cytotoxicity tests were carried on J774.2 macrophage cells using Glucantime as the reference drug. The mechanisms of action were analysed performing metabolite excretion and transmission electronic microscope ultrastructural alteration studies. RESULTS: Nine flavonoids showed leishmanicidal activity against promastigote as well as amastigote forms of Leishmania infantum and L. braziliensis. These compounds were nontoxic to mammalian cells and were effective at similar concentrations up to or lower than that of the reference drug (Glucantime). The results showed that 2(″)-acetylpetiolaroside (compound 8) was clearly the most active. CONCLUSION: This study has demonstrated that flavonoid derivatives are active against L. infantum and L. braziliensis.

Gallic Acid: A Natural Phenolic Compound Exerting Antitumoral Activities in Colorectal Cancer via Interaction with G-Quadruplexes.

Natural phenolic compounds have gained momentum for the prevention and treatment of cancer, but their antitumoral mechanism of action is not yet well understood. In the present study, we screened the antitumoral potential of several phenolic compounds in a cellular model of colorectal cancer (CRC). We selected gallic acid (GA) as a candidate in terms of potency and selectivity and extensively evaluated its biological activity. We report on the role of GA as a ligand of DNA G-quadruplexes (G4s), explaining several of its antitumoral effects, including the transcriptional inhibition of ribosomal and CMYC genes. In addition, GA shared with other established G4 ligands some effects such as cell cycle arrest, nucleolar stress, and induction of DNA damage. We further confirmed the antitumoral and G4-stabilizing properties of GA using a xenograft model of CRC. Finally, we succinctly demonstrate that GA could be explored as a therapeutic agent in a patient cohort with CRC. Our work reveals that GA, a natural bioactive compound present in the diet, affects gene expression by interaction with G4s both in vitro and in vivo and paves the way towards G4s targeting with phenolic compounds.

Biological activity of three novel complexes with the ligand 5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one against Leishmania spp.

OBJECTIVES: We report on new 1,2,4-triazolo[1,5-a]pyrimidine complexes that have been developed and examined for both antiproliferative in vitro activity against Leishmania infantum and Leishmania braziliensis, and report their possible mechanism of action on L. infantum and L. braziliensis. RESULTS: Antileishmanial effects are described for newly synthesized Cu(II) and Co(II) complexes containing 5-methyl-1,2,4-triazolo[1,5-a]pyrimidin-7(4H)-one (HmtpO) as a ligand. These complexes display a wide structural diversity: (i) mononuclear unit, [Cu(HmtpO)2(H2O)3](ClO4)2·H2O (1); (ii) two-dimensional framework, {[Cu(HmtpO)2(H2O)2](ClO4)(2)·2HmtpO}n (2); and (iii) chains, {[Co(HmtpO)(H2O)3](ClO4)(2)·2H2O}n (3). Compounds 1 and 2 appeared to be the most active against L. infantum (IC50 20.0 and 24.4 µM, respectively), and compounds 1 and 3 the most active against L. braziliensis (IC50 22.1 and 23.5 µM, respectively), with IC50s similar to those of the reference drug, glucantime (18.0 µM for L. infantum and 25.6 µM for L. braziliensis). These compounds were not toxic towards J774.2 macrophages. IC25 decreased infection capacity and severely reduced the multiplication of intracellular amastigotes, following the trend 1>3>2 for L. infantum and 3>1>2 for L. braziliensis. These complexes had an effect on the energy metabolism of the parasites at the level of the NAD+/NADH balance and the organelle membranes, causing their degradation and cell death. CONCLUSIONS: Cellular proliferation, metabolic and ultrastructural studies showed that the compounds 2>1>3 were highly active against L. infantum and L. braziliensis.

In vitro and in vivo trypanocidal activity of flavonoids from Delphinium staphisagria against Chagas disease.

The in vitro and in vivo trypanocidal activities of nine flavonoids (1-9) isolated from the aerial parts of Delphinium staphisagria have been studied in both the acute and chronic phases of Chagas disease. The antiproliferative activity of these substances against Trypanosoma cruzi (epimastigote, amastigote, and trypomastigote forms) in some cases exhibited more potent antitrypanosomatid activity and lower toxicity than the reference drug, benznidazole. Studies in vitro using ultrastructural analysis together with metabolism-excretion studies were also performed in order to identify the possible action mechanism of the compounds tested. Alterations mainly at the level of the mitochondria may explain metabolic changes in succinate and acetate production, perhaps due to the disturbance of the enzymes involved in sugar metabolism within the mitochondrion. In vivo studies provided results consistent with those observed in vitro. No signs of toxicity were detected in mice treated with the flavonoids tested, and the parasitic charge was significantly lower than in the control assay with benznidazole. The effects of these compounds were also demonstrated with the change in the anti-T. cruzi antibody levels during the chronic stage.

Gut Microbiota and the Quality of Oral Anticoagulation in Vitamin K Antagonists Users: A Review of Potential Implications.

The efficacy and safety of vitamin K antagonists (VKAs) as oral anticoagulants (OACs) depend on the quality of anticoagulation control, as reflected by the mean time in therapeutic range (TTR). Several factors may be involved in poor TTR such as comorbidities, high inter-individual variability, interacting drugs, and non-adherence. Recent studies suggest that gut microbiota (GM) plays an important role in the pathogenesis of cardiovascular diseases, but the effect of the GM on anticoagulation control with VKAs is unknown. In the present review article, we propose different mechanisms by which the GM could have an impact on the quality of anticoagulation control in patients taking VKA therapy. We suggest that the potential effects of GM may be mediated first, by an indirect effect of metabolites produced by GM in the availability of VKAs drugs; second, by an effect of vitamin K-producing bacteria; and finally, by the structural modification of the molecules of VKAs. Future research will help confirm these hypotheses and may suggest profiles of bacterial signatures or microbial metabolites, to be used as biomarkers to predict the quality of anticoagulation. This could lead to the design of intervention strategies modulating gut microbiota, for example, by using probiotics.

Treatment strategies for patients with atrial fibrillation and anticoagulant-associated intracranial hemorrhage: an overview of the pharmacotherapy.

INTRODUCTION: Oral anticoagulants (OAC) reduce stroke/systemic embolism and mortality risks in atrial fibrillation (AF). However, there is an inherent bleeding risk with OAC, where intracranial hemorrhage (ICH) is the most feared, disabling, and lethal complication of this therapy. Therefore, the optimal management of OAC-associated ICH is not well defined despite multiple suggested strategies. AREAS COVERED: In this review, the authors describe the severity and risk factors for OAC-associated ICH and the associated implications for using DOACs in AF patients. We also provide an overview of the management of OAC-associated ICH and treatment reversal strategies, including specific and nonspecific reversal agents as well as a comprehensive summary of the evidence about the resumption of DOAC and the optimal timing. EXPERT OPINION: In the setting of an ICH, supportive care/measures are needed, and reversal of anticoagulation with specific agents (including administration of vitamin K, prothrombin complex concentrates, idarucizumab and andexanet alfa) should be considered. Most patients will likely benefit from restarting anticoagulation after an ICH and permanently withdrawn of OAC is associated with worse clinical outcomes. Although the timing of OAC resumption is still under debate, reintroduction after 4-8 weeks of the bleeding event may be possible, after a multidisciplinary approach to decision-making.

The Use of Biomarkers in Clinical Management Guidelines: A Critical Appraisal.

In cardiovascular disease (CVD), biomarkers (i.e., "biological markers") could have multiple roles in understanding the complexity of cardiovascular (CV) pathophysiology and to offer an integrated approach to management. Biomarkers could help in daily practice as a diagnostic tool, to monitor therapy response, to assess prognosis and as early marker of CV damage, or to stratify risk. In recent years, the role of biomarkers in CVD is even more relevant and some have recently been included in clinical management guideline recommendations. The aim of this review is to discuss the recommendations in clinical guidelines of various biomarkers and to review their usefulness in daily clinical practice. Ultimately, a balance is needed between simplicity and practicality for clinical decision-making. Most biomarkers (whether blood, urine, or imaging-based) will improve on clinical risk stratification, but awaiting biomarker results may lead to delays in the initiation of therapy, for example, anticoagulation for stroke prevention in atrial fibrillation. Many biomarkers are nonspecific, being predictive of many CV and non-CV outcomes, so would be better as "rule-out" rather than "rule-in" assessments. Derivation of some biomarkers have also been made in highly selected clinical trial cohorts, where measurement is made at baseline but outcomes determined many years later; given the dynamic nature of risk in the "real world" where patients get older and develop incident risk factors, this may give a false impression of the risk profile. Finally, some laboratory biomarkers have a diurnal variation and inter-/intravariability (and lower limits of detection) in assays, which may be expensive, are added considerations.

Evolutionary cell biology traces the rise of the exomer complex in Fungi from an ancient eukaryotic component.

Cargo is transported from the trans-Golgi Network to the plasma membrane by adaptor complexes, which are pan-eukaryotic components. However, in yeast, cargo can also be exported by the exomer complex, a heterotetrameric protein complex consisting of two copies of Chs5, and any two members of four paralogous proteins (ChAPs). To understand the larger relevance of exomer, its phylogenetic distribution and function outside of yeast need to be explored. We find that the four ChAP proteins are derived from gene duplications after the divergence of Yarrowia from the remaining Saccharomycotina, with BC8 paralogues (Bch2 and Chs6) being more diverged relative to the BB8 paralogues (Bch1 and Bud7), suggesting neofunctionalization. Outside Ascomycota, a single preduplicate ChAP is present in nearly all Fungi and in diverse eukaryotes, but has been repeatedly lost. Chs5, however, is a fungal specific feature, appearing coincidentally with the loss of AP-4. In contrast, the ChAP protein is a wide-spread, yet uncharacterized, membrane-trafficking component, adding one more piece to the increasingly complex machinery deduced as being present in our ancient eukaryotic ancestor.