Aspergillus tubingensis Endocarditis: A Case Report and Review of the Literature

Mycopathologia - Aspergillus endocarditis is a rare infection that may affect immunocompetent patients following heart valve replacement or heart surgery. We report the case of a 39 year...     

Unraveling interactions of cell cycle-regulating proteins Sic1 and B-type cyclins in living yeast cells: a FLIM-FRET approach

Sic1, cyclin-dependent kinase inhibitor of budding yeast, is synthesized in anaphase and largely degraded at the S-phase onset to regulate timing of DNA synthesis. Sic1 interacts with phase-specific B-type cyclin (Clb)-kinase (Cdk1) complexes, central regulators in cell cycle control. Its appearance is timed to mediate reduction in kinase activities at appropriate stages. Clbs are unstable proteins with extremely short half-lives. Interactions of Sic1 with Clbs have been detected both in vitro and in vivo by high-throughput genome-wide screenings. Furthermore, we have recently shown that Sic1 regulates waves of Clbs, acting as a timer in their appearance, thus controlling Cdk1-Clbs activation. The molecular mechanism is not yet fully understood but is hypothesized to occur via stoichiometric binding of Sic1 to Cdk1-Clb complexes. Using F?rster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy (FLIM), we showed association of Sic1 to Clb cyclins in living yeast cells. This finding is consistent with the notion that inhibition of kinase activity can occur over the whole cell cycle progression despite variable Sic1 levels. Specifically, Sic1/Clb3 interaction was observed for the first time, and Sic1/Clb2 and Sic1/Clb5 pairs were confirmed, but no Sic1/Clb4 interaction was found, which suggests that, despite high functional homology between Clbs, only some of them can target Sic1 function in vivo.     

Genome-wide evaluation of histone methylation changes associated with leaf senescence in Arabidopsis

Leaf senescence is the orderly dismantling of older tissue that allows recycling of nutrients to developing portions of the plant and is accompanied by major changes in gene expression. Histone modifications correlate to levels of gene expression, and this study utilizes ChIP-seq to classify activating H3K4me3 and silencing H3K27me3 marks on a genome-wide scale for soil-grown mature and naturally senescent Arabidopsis leaves. ChIPnorm was used to normalize data sets and identify genomic regions with significant differences in the two histone methylation patterns, and the differences were correlated to changes in gene expression. Genes that showed an increase in the H3K4me3 mark in older leaves were senescence up-regulated, while genes that showed a decrease in the H3K4me3 mark in the older leaves were senescence down-regulated. For the H3K27me3 modification, genes that lost the H3K27me3 mark in older tissue were senescence up-regulated. Only a small number of genes gained the H3K27me3 mark, and these were senescence down-regulated. Approximately 50% of senescence up-regulated genes lacked the H3K4me3 mark in both mature and senescent leaf tissue. Two of these genes, SAG12 and At1g73220, display strong senescence up-regulation without the activating H3K4me3 histone modification. This study provides an initial epigenetic framework for the developmental transition into senescence.     

Sic1 as a timer of Clb cyclin waves in the yeast cell cycle - design principle of not just an inhibitor

Cellular systems biology aims to uncover design principles that describe the properties of biological networks through interaction of their components in space and time. The cell cycle is a complex system regulated by molecules that are integrated into functional modules to ensure genome integrity and faithful cell division. In budding yeast, cyclin-dependent kinases (Cdk1/Clb) drive cell cycle progression, being activated and inactivated in a precise temporal sequence. In this module, which we refer to as the 'Clb module', different Cdk1/Clb complexes are regulated to generate waves of Clb activity, a functional property of cell cycle control. The inhibitor Sic1 plays a critical role in the Clb module by binding to and blocking Cdk1/Clb activity, ultimately setting the timing of DNA replication and mitosis. Fifteen years of research subsequent to the identification of Sic1 have lead to the development of an integrative approach that addresses its role in regulating the Clb module. Sic1 is an intrinsically disordered protein and achieves its inhibitory function by cooperative binding, where different structural regions stretch on the Cdk1/Clb surface. Moreover, Sic1 promotes S?phase entry, facilitating Cdk1/Clb5 nuclear transport, and therefore revealing a double function of inhibitor/activator that rationalizes a mechanism to prevent precocious DNA replication. Interestingly, the investigation of Clb temporal dynamics by mathematical modelling and experimental validation provides evidence that Sic1 acts as a timer to coordinate oscillations of Clb cyclin waves. Here we review these findings, focusing on the design principle underlying the Clb module, which highlights the role of Sic1 in regulating phase-specific Cdk1/Clb activities.     

Targeting TNF-α to neoangiogenic vessels enhances lymphocyte infiltration in tumors and increases the therapeutic potential of immunotherapy

Abnormal tumor vasculature impairs T lymphocyte adhesion to endothelial cells and lymphocyte extravasation into neoplastic tissues, limiting the therapeutic potential of both active and adoptive immunotherapies. We have found that treatment of tumor-bearing mice with NGR-TNF, a Cys-Asn-Gly-Arg-Cys peptide-TNF fusion product capable of altering the endothelial barrier function and improving drug penetration in tumors, associated with the intratumor upregulation of leukocyte-endothelial cell adhesion molecules, the release of proinflammatory cytokines and chemokines, and the infiltration of tumor-specific effector CD8(+) T cells. As a result, NGR-TNF enhanced the therapeutic activity of adoptive and active immunotherapy, delaying tumor growth and prolonging survival. Furthermore, we have found that therapeutic effects of these combinations can be further increased by the addition of chemotherapy. Thus, these findings might be relevant for the design of novel immunotherapeutic approaches for cancer patients.     

Cyr61 activates retinal cells and prolongs photoreceptor survival in rd1 mouse model of retinitis pigmentosa

Subretinal injections with glial cell line‐derived neurotrophic factor (GDNF) rescue morphology as well as function of rod cells in mouse and rat animal models of retinitis pigmentosa. At the same time, it is postulated that this effect is indirect, mediated by activation of retinal Müller glial (RMG) cells. Here, we show that Cyr61/CCN1, one of the secreted proteins up‐regulated in primary RMG after glial cell line‐derived neurotrophic factor stimulation, provides neuroprotective and pro‐survival capacities: Recombinant Cyr61 significantly reduced photoreceptor (PR) cells death in organotypic cultures of Pde6b^rd1 retinas. To identify stimulated pathways in the retina, we treated Pde6b^rd1 retinal explants with Cyr61 and observed an overall increase in activated Erk1/2 and Stat3 signalling molecules characterized by activation‐site‐specific phosphorylation. To identify Cyr61 retinal target cells, we isolated primary porcine PR, RMG and retinal pigment epithelium (RPE) cells and exposed them separately to Cyr61. Here, RMG as well as RPE cells responded with induced phosphorylation of Erk1/2, Stat3 and Akt. In PR, no increase in phosphorylation in any of the studied proteins was detected, suggesting an indirect neuroprotective effect of Cyr61. Cyr61 may thus act as an endogenous pro‐survival factor for PR, contributing to the complex repertoire of neuroprotective activities generated by RMG and RPE cells.

     

Light-Harvesting Complex Protein LHCBM9 Is Critical for Photosystem II Activity and Hydrogen Production in Chlamydomonas reinhardtii

Photosynthetic organisms developed multiple strategies for balancing light-harvesting versus intracellular energy utilization to survive ever-changing environmental conditions. The light-harvesting complex (LHC) protein family is of paramount importance for this function and can form light-harvesting pigment protein complexes. In this work, we describe detailed analyses of the photosystem II (PSII) LHC protein LHCBM9 of the microalga Chlamydomonas reinhardtii in terms of expression kinetics, localization, and function. In contrast to most LHC members described before, LHCBM9 expression was determined to be very low during standard cell cultivation but strongly increased as a response to specific stress conditions, e.g., when nutrient availability was limited. LHCBM9 was localized as part of PSII supercomplexes but was not found in association with photosystem I complexes. Knockdown cell lines with 50 to 70% reduced amounts of LHCBM9 showed reduced photosynthetic activity upon illumination and severe perturbation of hydrogen production activity. Functional analysis, performed on isolated PSII supercomplexes and recombinant LHCBM9 proteins, demonstrated that presence of LHCBM9 resulted in faster chlorophyll fluorescence decay and reduced production of singlet oxygen, indicating upgraded photoprotection. We conclude that LHCBM9 has a special role within the family of LHCII proteins and serves an important protective function during stress conditions by promoting efficient light energy dissipation and stabilizing PSII supercomplexes.     

Are atopy and eosinophilic bronchial inflammation associated with relapsing forms of chronic rhinosinusitis with nasal polyps?

Background

The aetiopathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) is still unknown. The role of atopy and the concept of united airways in such patients are still a matter of debate. In this pilot study we aimed at evaluating the degree of eosinophilic inflammation and the frequency of atopy in a cohort of CRSwNP patients candidate for Functional Endoscopic Sinus Surgery (FESS) and assessing the association between these factors and relapsing forms of CRSwNP.

Methods

30 patients (18 men, 12 women) with CRSwNP eligible for FESS were evaluated before and after surgery. Preoperative investigation included: history of previous relapse after FESS, clinical and laboratory allergologic assessment, spirometry, methacholine challenge, blood eosinophilia and determination of the fraction of nitric oxide in exhaled air (FeNO). Nasal fibroendoscopy, spirometry and FeNO determination were also assessed prospectively at 3 and 27 months post-FESS.

Results

18/30 subjects were atopic, 6/18 (33 %) were monosensitized, 16/30 (53 %) were asthmatics and 10/30 (33 %) had non steroidalantinflammatory drugs (NSAIDs) hypersensitivity. Twenty-one patients (70 %) were classified as relapsers, 15/18 (83 %) among atopics, 6/12 (50 %) among non atopics (p = 0.05). Among patients with NSAIDs hypersensitivity, 9/10 (90 %) were relapsers. The median IgE concentration was 161.5 UI/mL in relapsers and 79 UI/mL in non-relapsers (ns). The mean FeNO decreased after FESS (43.1–26.6 ppb) in 84 % of patients, but this effect disappeared over time (FeNO = 37.7 ppb at 27 months). Higher levels of FeNO pre-FESS were detected in atopics, and in particular in relapsing ones (median 51.1 ppb vs 22.1, ns). Higher levels of FeNO pre-FESS were detected in asthmatic patients, especially in those who relapsed (median: 67 vs 64.85 ppb in non-relapsed patients, ns). The Tiffeneau Index (FEV1/FVC) was significantly lower in asthmatic relapsers than in non relapsers asthmatics (94.7 ± 11.1 versus 105 ± 5.9—p = 0.04). Patients with asthma and atopy had a major risk of relapse (p = 0.05).

Conclusion

In our pilot study, atopy, severe asthma, bronchial inflammation, NSAIDs hypersensitivity and high level of total IgE are possible useful prognostic factors for the proneness to relapse after FESS. The role of allergy in CRSwNP pathogenesis should consequently be given deeper consideration. Allergen specific immunotherapy, combined with anti-IgE therapy, may have an immunomodulatory effect preventing polyps relapse and need to be investigated.

Electronic supplementary material

The online version of this article (doi:10.1186/s12948-015-0026-8) contains supplementary material, which is available to authorized users.     

Optimized synthesis and characterization of N-acylethanolamines and O-acylethanolamines, important family of lipid-signalling molecules

The endocannabinoid anandamide (N-arachidonoylethanolamine, AEA), a physiologically occurring bioactive compound on CB1 and CB2 receptors, has multiple physiological functions. Since the discovery of AEA additional non-cannabinoid endogenous compounds such as N-palmitoylethanolamine (PEA), and N-oleoylethanolamine (OEA) have been identified from mammalian tissues. Virodhamine (O-arachidonoylethanolamine, VA) is the only identified new member of the endocannabinoid family that is characterised by an ester linkage between acylic acid and ethanolamine instead of the amide linkage found in AEA and others non-cannabinoid N-acylethanolamines. It has been reported, as a cautionary note for lipid analyses, that VA can be produced nonenzymatically from AEA (and vice versa) as consequence of O,N-acyl migrations. O,N-acyl migrations are well documented in synthetic organic chemistry literature, but are not well described or recognized with regard to methods in lipid isolation or lipid enzyme studies. We here report an economical and effective protocol for large scale synthesis and characterization of some N- and O-acylethanolamines that could be useful as reference standards in order to investigate their possible formation in biological membranes, with potentially interesting biological properties.