Breakthrough pulmonary Aspergillus fumigatus infection with multiple triazole resistance in a Spanish patient with chronic myeloid leukemia

BackgroundAn allogeneic hematopoietic cell transplantation (allo-HCT) patient presented with chronic pulmonary aspergillosis associated to pulmonary graft versus host disease (GVHD) and was treated for a long time with several antifungal agents that were administered as prophylaxis, combination therapies, and maintenance treatment. The patient suffered from a breakthrough invasive pulmonary aspergillosis due to Aspergillus fumigatus after long-term antifungal therapy.Material and methodsSeveral isolates were analyzed. First isolates were susceptible in vitro to all azole agents. However, after prolonged treatment with itraconazole and voriconazole a multiple azole resistant A. fumigatus isolate was cultured from bronchoalveolar lavage (BAL) when the patient was suffering from an invasive infection, and cavitary lesions were observed.ResultsAnalysis of the resistant mechanisms operating in the last strain led us to report the first isolation in Spain of an azole resistant A. fumigatus strain harboring the L98H mutation in combination with the tandem repeat (TR) alteration in CYP51A gene (TR-L98H). Long-term azole therapy may increase the risk of resistance selecting strains exhibiting reduced susceptibility to these compounds. However, since the isolates were genetically different the suggestion that could be made is that the resistance was not induced during the prolonged azole therapy but the patient might simply have acquired this resistant isolate from the environment, selected by the therapy.ConclusionsThese findings suggest that in all long-term treatments with antifungal agents, especially with azoles, repeated sampling and regular susceptibility testing of strains isolated is necessary as resistant isolates could be selected.     

Review ArticleNaphthalene Combretastatin Analogues: Synthesis,Cytotoxicity and Antitubulin Activity

Synthesis and evaluation of new combretastatin analogues with varied modifications on the bridge and the aromatic rings, have shown that the 2-naphthyl moiety is a good surrogate for the 3-hydroxy-4-methoxyphenyl (B-ring) of combretastatin A-4. Other bicyclic systems, such as 6(7)-quinolyl and 5-indolyl, can replace the B-ring, but they produce less potent analogues in the cytotoxicity and tubulin polymerization inhibition assays. Other modifications are detrimentral for the potency of the studied analogues. The 2-naphthyl combretastatin 53 and the related 6-quinolyl combretastatin 106 analogues are the most potent among the derivatives of this type, whereas 92 and 95 are the most potent among the naphthalene derivatives with a heterocycle in the bridge. Previous and new results in this family of combretastatin analogues are discussed.     

Increased dosage of Ink4/Arf protects against glucose intolerance and insulin resistance associated with aging

Recent genome‐wide association studies have linked type‐2 diabetes mellitus to a genomic region in chromosome 9p21 near the Ink4/Arf locus, which encodes tumor suppressors that are up‐regulated in a variety of mammalian organs during aging. However, it is unclear whether the susceptibility to type‐2 diabetes is associated with altered expression of the Ink4/Arf locus. In the present study, we investigated the role of Ink4/Arf in age‐dependent alterations of insulin and glucose homeostasis using Super‐Ink4/Arf mice which bear an extra copy of the entire Ink4/Arf locus. We find that, in contrast to age‐matched wild‐type controls, Super‐Ink4/Arf mice do not develop glucose intolerance with aging. Insulin tolerance tests demonstrated increased insulin sensitivity in Super‐Ink4/Arf compared with wild‐type mice, which was accompanied by higher activation of the insulin receptor substrate (IRS)‐PI3K‐AKT pathway in liver, skeletal muscle and heart. Glucose uptake studies in Super‐Ink4/Arf mice showed a tendency toward increased ¹⁸F‐fluorodeoxyglucose uptake in skeletal muscle compared with wild‐type mice (P = 0.079). Furthermore, a positive correlation between glucose uptake and baseline glucose levels was observed in Super‐Ink4/Arf mice (P < 0.008) but not in wild‐type mice. Our studies reveal a protective role of the Ink4/Arf locus against the development of age‐dependent insulin resistance and glucose intolerance.     

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

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

Comparison of PCR protocols for detecting Histoplasma capsulatum DNA through a multicenter study

BackgroundA multicenter study was conducted. A panel containing DNA from Histoplasma capsulatum, as well as negative and cross-reaction controls, was sent to five different laboratories, members of the MICOMOL network from CYTED Program.AimsThe objective was to assess the accuracy of different PCR protocols to detect H. capsulatum DNA.MethodsSeven different PCR protocols were tested. They were based on PCR techniques and used unicopy and multicopy targets.ResultsMost of these protocols (4/7) were able to detect the smallest amounts of fungal DNA (10² fg/μl). Overall sensitivity was 86% and specificity was 100%. The protocol based on a unicopy target (SCAR₂₂₀) presented lower sensitivity (43%) but 100% specificity. The real-time protocols tested were highly reproducible, sensitive, and specific. Neither false positives nor cross-reactions were detected in any protocol.ConclusionsAll laboratories were able to amplify H. capsulatum DNA, and real-time PCR seems to be a promising tool to efficiently detect this pathogen in clinical samples.     

Effect of lead stress on mineral content and growth of wheat (Triticum aestivum) and spinach (Spinacia oleracea) seedlings

Lead (Pb) is the most common heavy metal contaminant in the environment. Pb is not an essential element for plants, but they absorb it when it is present in their environment, especially in rural areas when the soil is polluted by automotive exhaust and in fields contaminated with fertilizers containing heavy metal impurities. To investigate lead effects on nutrient uptake and metabolism, two plant species, spinach (Spinacia oleracea) and wheat (Triticum aestivum), were grown under hydroponic conditions and stressed with lead nitrate, Pb(NO₃)₂, at three concentrations (1.5, 3, and 15 mM).Lead is accumulated in a dose-dependent manner in both plant species, which results in reduced growth and lower uptake of all mineral ions tested. Total amounts and concentrations of most mineral ions (Na, K, Ca, P, Mg, Fe, Cu and Zn) are reduced, although Mn concentrations are increased, as its uptake is reduced less relative to the whole plant’s growth. The deficiency of mineral nutrients correlates in a strong decrease in the contents of chlorophylls a and b and proline in both species, but these effects are less pronounced in spinach than in wheat. By contrast, the effects of lead on soluble proteins differ between species; they are reduced in wheat at all lead concentrations, whereas they are increased in spinach, where their value peaks at 3 mM Pb.The relative lead uptake by spinach and wheat, and the different susceptibility of these two species to lead treatment are discussed.     

De Novo Assembly and Functional Annotation of the Olive (Olea europaea) Transcriptome

Olive breeding programmes are focused on selecting for traits as short juvenile period, plant architecture suited for mechanical harvest, or oil characteristics, including fatty acid composition, phenolic, and volatile compounds to suit new markets. Understanding the molecular basis of these characteristics and improving the efficiency of such breeding programmes require the development of genomic information and tools. However, despite its economic relevance, genomic information on olive or closely related species is still scarce. We have applied Sanger and 454 pyrosequencing technologies to generate close to 2 million reads from 12 cDNA libraries obtained from the Picual, Arbequina, and Lechin de Sevilla cultivars and seedlings from a segregating progeny of a Picual × Arbequina cross. The libraries include fruit mesocarp and seeds at three relevant developmental stages, young stems and leaves, active juvenile and adult buds as well as dormant buds, and juvenile and adult roots. The reads were assembled by library or tissue and then assembled together into 81 020 unigenes with an average size of 496 bases. Here, we report their assembly and their functional annotation.     

Eye trematode infection in small passerines in Peru caused by Philophthalmus lucipetus,an agent with a zoonotic potential spread by an invasive freshwater snail

Until now, four species of eye trematodes have been found in South America. Of them, Philophthalmus lucipetus (synonymized with Philophthalmus gralli) displays a broad host spectrum, with at least 30 bird species (prevalently large water birds), five mammal species and humans serving as definitive hosts, and with snails Fagotia (Microcolpia) acicularis, Amphimelania holandri, Melanopsis praemorsa and Melanoides tuberculata serving as intermediate hosts. When examining a total of 50 birds of ten species in the wetland of Pantanos de Villa, Lima, Peru in July 2011, eye trematodes were identified visually in the edematous conjunctival sac of 11 (48%) out of 23 resident many-colored rush tyrants Tachuris rubrigastra. Based on morphometric characteristics, the trematodes were identified as P. lucipetus. ITS2 and CO1 gene of the examined specimens combined showed a 99% similarity to an Iranian isolate of Philophthalmus sp. from the intermediate host Melanoides tuberculata, an invasive freshwater snail, suggesting that these two isolates represent the same species with a wide geographical range. Moreover, the prevalence of infection with the philophthalmid cercariae was 31% in 744 Melanoides tuberculata examined in Pantanos de Villa in 2010. It is evident that P. lucipetus occurs throughout the world as well as locally, including Eurasia and South America. Here we report this trematode for the first time in Peru, and we were the first to sequence any of the South American eye trematodes. Low host specificity of P. lucipetus and the invasive character of Melanoides tuberculata as a competent intermediate host suggest that eye trematodosis caused by P. lucipetus may emerge frequently in various parts of the world, especially in the tropics. Increase of the zoonotic potential of the P. lucipetus associated with this invasive snail spreading across the world is predictable and should be of interest for further research.