Sensing and Responding to Hypersaline Conditions and the HOG Signal Transduction Pathway in Fungi Isolated from Hypersaline Environments: and .

Sensing and responding to changes in NaCl concentration in hypersaline environments is vital for cell survival. In this paper, we identified and characterized key components of the high-osmolarity glycerol (HOG) signal transduction pathway, which is crucial in sensing hypersaline conditions in the extremely halotolerant black yeast and in the obligate halophilic fungus . Both organisms were isolated from solar salterns, their predominating ecological niche.

Electron Paramagnetic Resonance Gives Evidence for the Presence of Type 1 Gonadotropin-Releasing Hormone Receptor (GnRH-R) in Subdomains of Lipid Rafts.

This study investigated the effect of type 1 gonadotropin releasing hormone receptor (GnRH-R) localization within lipid rafts on the properties of plasma membrane (PM) nanodomain structure. Confocal microscopy revealed colocalization of PM-localized GnRH-R with GM-enriched raft-like PM subdomains. Electron paramagnetic resonance spectroscopy (EPR) of a membrane-partitioned spin probe was then used to study PM fluidity of immortalized pituitary gonadotrope cell line αT3-1 and HEK-293 cells stably expressing GnRH-R and compared it with their corresponding controls (αT4 and HEK-293 cells).

Enrichment of plasma extracellular vesicles for reliable quantification of their size and concentration for biomarker discovery.

Human plasma is a complex fluid, increasingly used for extracellular vesicle (EV) biomarker studies. Our aim was to find a simple EV-enrichment method for reliable quantification of EVs in plasma to be used as biomarker of disease. Plasma of ten healthy subjects was processed using sedimentation rate- (sucrose cushion ultracentrifugation-sUC) and size- (size exclusion chromatography-SEC) based methods.

PKH26 labeling of extracellular vesicles: Characterization and cellular internalization of contaminating PKH26 nanoparticles.

PKH lipophilic dyes are highly fluorescent and stain membranes by intercalating their aliphatic portion into the exposed lipid bilayer. They have established use in labeling and tracking of cells in vivo and in vitro. Despite wide use of PKH-labeled extracellular vesicles (EVs) in cell targeting and functional studies, nonEV-associated fluorescent structures have never been examined systematically, nor was their internalization by cells.

Strategies of adaptation of microorganisms of the three domains of life to high salt concentrations.

Hypersaline environments with salt concentrations up to NaCl saturation are inhabited by a great diversity of microorganisms belonging to the three domains of life. They all must cope with the low water activity of their environment, but different strategies exist to provide osmotic balance of the cells' cytoplasm with the salinity of the medium. One option used by many halophilic Archaea and a few representatives of the Bacteria is to accumulate salts, mainly KCl and to adapt the entire intracellular machinery to function in the presence of molar concentrations of salts.

Viral protein Nef is detected in plasma of half of HIV-infected adults with undetectable plasma HIV RNA.

Objective: To address the role of translationally active HIV reservoir in chronic inflammation and non-AIDS related disorders, we first need a simple and accurate assay to evaluate viral protein expression in virally suppressed subjects.

Design: We optimized an HIV Nef enzyme-linked immunosorbent assay (ELISA) and used it to quantify plasma Nef levels as an indicator of the leaky HIV reservoir in an HIV-infected cohort.

Methods: This study accessed 134 plasma samples from a well-characterized cohort study of HIV-infected and uninfected adults in San Francisco (the SCOPE cohort).

Trans-Activation Response Element RNA is Detectable in the Plasma of a Subset of Aviremic HIV-1-Infected Patients.

Determining the HIV-1 reservoir size in infected individuals is of great importance for improvement of their treatment. Plasma trans-activation response element (TAR) RNA has been suggested as one of the possible biomarkers. TAR RNA is produced during non-processive transcription in HIV-1 productively infected and latent T cells.

Nef is secreted in exosomes from Nef.GFP-expressing and HIV-1-infected human astrocytes.

HIV-1 infection of the central nervous system causes HIV-associated neurocognitive disorders, even in aviremic patients. Although astrocyte malfunction was associated to these disorders, their implication is overshadowed by contributions of microglia and macrophages. Astrocytes are infected with HIV-1 in vivo and express a relevant amount of viral protein Nef.

Insight into the Recent Genome Duplication of the Halophilic Yeast : Combining an Improved Genome with Gene Expression and Chromatin Structure.

Extremophilic organisms demonstrate the flexibility and adaptability of basic biological processes by highlighting how cell physiology adapts to environmental extremes. Few eukaryotic extremophiles have been well studied and only a small number are amenable to laboratory cultivation and manipulation. A detailed characterization of the genome architecture of such organisms is important to illuminate how they adapt to environmental stresses.

Reconstruction of the High-Osmolarity Glycerol (HOG) Signaling Pathway from the Halophilic Fungus Wallemia ichthyophaga in Saccharomyces cerevisiae.

The basidiomycetous fungus Wallemia ichthyophaga grows between 1.7 and 5.1 M NaCl and is the most halophilic eukaryote described to date.

Transport Systems in Halophilic Fungi.

Fungi that tolerate very high environmental NaCl concentrations are good model systems to study mechanisms that enable them to endure osmotic and salinity stress. The whole genome sequences of six such fungal species have been analysed: Hortaea werneckii, Wallemia ichthyophaga and four Aureobasidium spp.: A.

Alcohol Dependence and Genetic Variability in the Serotonin Pathway among Currently and Formerly Alcohol-Dependent Males.

Background: Genes involved in the serotonin pathway may determine the susceptibility to alcohol dependence and its severity. The present study explored whether specific polymorphisms in the serotonin pathway could be associated with alcohol dependence or alcohol-related psychopathological symptoms.

Methods: The cohort comprised 101 currently and 100 formerly alcohol-dependent males, as well as 97 male healthy blood donors.

Genetic variability in tryptophan hydroxylase 2 gene in alcohol dependence and alcohol-related psychopathological symptoms.

Heritability plays an important role in the development and expression of alcohol dependence. The present genetic association study explored the role of TPH2 polymorphisms and their haplotypes to investigate its role in alcohol dependence and comorbid psychopathological symptoms. The sample included 101 subjects currently diagnosed as alcohol abusers, 100 abstinent alcohol-dependent subjects and 97 healthy controls.

The unique characteristics of HOG pathway MAPKs in the extremely halotolerant Hortaea werneckii.

HwHog1A/B, Hortaea werneckii homologues of the MAP kinase Hog1 from Saccharomyces cerevisiae, are vital for the extreme halotolerance of H. werneckii. In mesophilic S.

Genetic variability in CYP2E1 and catalase gene among currently and formerly alcohol-dependent male subjects.

Aims: The present study explored whether specific single-nucleotide polymorphisms in alcohol metabolic pathway are associated with alcohol dependence or alcohol-related psychopathological symptoms.

Methods: Three groups of male unrelated subjects were included: 101 currently alcohol-dependent patients, 100 formerly alcohol-dependent subjects and 97 healthy controls. The following questionnaires were implemented: AUDIT, Zung Depression and Anxiety scale, Brief Social Phobia Scale, Yale-Brown Obsessive Compulsive Scale, Obsessive Compulsive Drinking Scale and Buss-Durkee Hostility Inventory.

HwHog1 kinase activity is crucial for survival of Hortaea werneckii in extremely hyperosmolar environments.

Although suggested, the involvement of the HOG pathway in adaptation processes in extremely halotolerant fungus Hortaea werneckii has never been specifically demonstrated. Here, we show that the H. werneckii HOG pathway is very robust, and that it includes two functionally redundant MAPK homologues, HwHog1A and HwHog1B, that show osmolyte-type-dependent phosphorylation.

Adaptation to high salt concentrations in halotolerant/halophilic fungi: a molecular perspective.

Molecular studies of salt tolerance of eukaryotic microorganisms have until recently been limited to the baker's yeast Saccharomyces cerevisiae and a few other moderately halotolerant yeast. Discovery of the extremely halotolerant and adaptable fungus Hortaea werneckii and the obligate halophile Wallemia ichthyophaga introduced two new model organisms into studies on the mechanisms of salt tolerance in eukaryotes. H.

Insertion of a specific fungal 3'-phosphoadenosine-5'-phosphatase motif into a plant homologue improves halotolerance and drought tolerance of plants.

Soil salinity and drought are among the most serious agricultural and environmental problems of today. Therefore, investigations of plant resistance to abiotic stress have received a lot of attention in recent years. In this study, we identified the complete coding sequence of a 3'-phosphoadenosine-5'-phosphatase protein, ApHal2, from the halotolerant yeast Aureobasidium pullulans.

Whole genome duplication and enrichment of metal cation transporters revealed by de novo genome sequencing of extremely halotolerant black yeast Hortaea werneckii.

Hortaea werneckii, ascomycetous yeast from the order Capnodiales, shows an exceptional adaptability to osmotically stressful conditions. To investigate this unusual phenotype we obtained a draft genomic sequence of a H. werneckii strain isolated from hypersaline water of solar saltern.

Melanin is crucial for growth of the black yeast Hortaea werneckii in its natural hypersaline environment.

Melanin has an important role in the ability of fungi to survive extreme conditions, like the high NaCl concentrations that are typical of hypersaline environments. The black fungus Hortaea werneckii that has been isolated from such environments has 1,8-dihydroxynaphthalene-melanin incorporated into the cell wall, which minimises the loss of glycerol at low NaCl concentrations. To further explore the role of melanin in the extremely halotolerant character of H.

The HOG signal transduction pathway in the halophilic fungus Wallemia ichthyophaga: identification and characterisation of MAP kinases WiHog1A and WiHog1B.

The high-osmolarity glycerol (HOG) pathway is one of the several MAP kinase cascades in fungi. It is the main signal transduction system that is responsible for cellular stress responses, and has primarily been studied in the context of osmotic stress. In the present study, we provide the first insights into the HOG pathway of the obligatory halophilic basidiomycetous fungus Wallemia ichthyophaga, with the characterisation of its two Hog1-like kinases: WiHog1A and WiHog1B.

The mycobiota of the salterns.

Solar salterns are constructed as shallow multi-pond systems for the production of halite through evaporation of seawater. The main feature of salterns is the discontinuous salinity gradient that provides a range of well-defined habitats with increasing salinities, from moderate to hypersaline. These present one of the most extreme environments, because of the low levels of biologically available water and the toxic concentrations of ions.

Fungal adaptation to extremely high salt concentrations.

Hypersaline environments support substantial microbial communities of selected halotolerant and halophilic organisms, including fungi from various orders. In hypersaline water of solar salterns, the black yeast Hortaea werneckii is by far the most successful fungal representative. It has an outstanding ability to overcome the turgor loss and sodium toxicity that are typical for hypersaline environments, which facilitates its growth even in solutions that are almost saturated with NaCl.

Adaptation of the glycerol-3-phosphate dehydrogenase Gpd1 to high salinities in the extremely halotolerant Hortaea werneckii and halophilic Wallemia ichthyophaga.

We report the first identification and characterisation of the glycerol-3-phosphate dehydrogenase (GPD) genes from extremely halophilic fungi. The black ascomycetous yeast Hortaea werneckii and the non-melanised basidiomycetous fungus Wallemia ichthyophaga inhabit similar hypersaline environments, yet they have two different strategies of haloadaptation through Gpd1-regulated glycerol synthesis. The extremely halotolerant H.

Extremophilic yeasts: plasma-membrane fluidity as determinant of stress tolerance.

Our aim was to investigate the response of selected yeasts and yeast-like fungi from extreme environments to various temperatures at the level of their plasma membranes, in order to elucidate the connections between their plasma-membrane fluidity (measured by electron paramagnetic resonance spectroscopy - EPR), growth temperature range, stress tolerance, and ecological distribution. Although all studied fungi can be considered mesophilic according to their growth temperature profiles, their plasma-membrane fluidity indicated otherwise. Arctic yeast Rhodosporidium diobovatum could be classified as psychrotolerant due to its higher average membrane fluidity.