Large-Scale Chromosomal Changes and Associated Fitness Consequences in Pathogenic Fungi

Pathogenic fungi encounter many different host environments to which they must adapt rapidly to ensure growth and survival. They also must be able to cope with alterations in established niches during long-term persistence in the host. Many eukaryotic pathogens have evolved a highly plastic genome, and large-scale chromosomal changes including aneuploidy, and loss of heterozygosity (LOH) can arise under various in vitro and in vivo stresses. Both aneuploidy and LOH can arise quickly during a single cell cycle, and it is hypothesized that they provide a rapid, albeit imprecise, solution to adaptation to stress until better and more refined solutions can be acquired by the organism. While LOH, with the extreme case of haploidization in Candida albicans, can purge the genome from recessive lethal alleles and/or generate recombinant progeny with increased fitness, aneuploidy, in the absence or rarity of meiosis, can serve as a non-Mendelian mechanism for generating genomic variation.     

Current Antifungal Agents for Treatment of Central Nervous System Infections

Invasive fungal infections have been increasing in incidence worldwide, primarily due to the growing population of immunosuppressed patients. Fungal central nervous system infections carry a high rate of morbidity and mortality due to difficulty in diagnosis and treatment. This paper provides a review of antifungal agents currently available in the United States, highlighting the pros and cons of each medication in the treatment of CNS infections. Investigational treatment approaches with combination therapy and iron chelation for CNS infections are also discussed. Despite our many advances in modern medicine, we still lack highly effective therapeutic options for treatment of cerebral infections with the angioinvasive fungi Aspergillus and the zygomycetes.     

Cardiotoxicity Induced by Antifungal Drugs

This review addresses the potential of antifungal drugs to cause cardiac toxicity. Many antifungal drugs, especially antifungal azoles, rarely cause torsades de pointes (TdP) and carry the risk of sudden death. Interventions to avoid TdP should include cautious use of azoles in combination with other drugs that cause QTc prolongation, and elimination of risk factors for TdP whenever possible. These risk factors include: hypokalemia, hypomagnesemia, severe bradycardia, and preexisting long QT syndrome. ECG monitoring should be considered when the use of multiple QT-prolonging drugs is unavoidable and TdP risk factors cannot be resolved. Itraconazole exhibits negative inotropic activity which may present as worsening heart failure in patients with preexisting heart failure. A few cases of severe bradycardia have also been described with voriconazole. Most cases of cardiac toxicity associated with amphotericin B are due to severe electrolyte abnormalities, rapid administration or overdose. Although cardiac toxicity is not common with the use of antifungal drugs, recognition of the potential to cause serious cardiac-related outcomes, evaluation of risk factors, and monitoring is warranted.     

Amphotericin B: How Much Is Enough?

The total curative dose of amphotericin B for any given fungal infection or specific patient is not precisely known. Prior to the availability of lipid formulations of amphotericin B (LFAB), dosing of amphotericin B was dictated by its associated toxicity. The unique pharmacokinetic features of each LFAB have led to differences in their recommended doses. Most published data have evaluated doses of 3–5 mg/kg/day, although niches exist for both higher and lower doses. Low-dose LFAB allows for intravenous broad-spectrum antifungal coverage without drug-drug interactions and with reduced toxicity. High-dose LFAB demonstrates increased fungal clearance in animal models, although this has not translated to improved clinical outcomes for most invasive fungal infections. Although available data do not demonstrate significant benefit associated with high-dose therapy, for liposomal amphotericin B, the data also demonstrate no significant harm. As such, the use of high-dose liposomal amphotericin B for salvage therapy may be a consideration.     

Essential Metals in Cryptococcus neoformans: Acquisition and Regulation

Transition metals such as iron, zinc, and copper play key roles in numerous biological processes, including innate immunity in vertebrates. In fact, control of the availability of these essential metals is an important component of the immune response to limit the growth of pathogens during infection. In turn, pathogens must overcome the extreme limitation of essential metals and potential metal toxicity to successfully cause disease. Recent work illuminates the mechanisms by which the pathogenic fungus Cryptococcus neoformans manages iron, copper, and zinc availability in support of proliferation in vertebrate hosts. In particular, the fungus makes use of high-affinity metal transport systems and complex regulatory networks that play critical roles in the virulence of the fungus.     

The Origin and Evolution of Vertebrate Glycine Transporters

In the vertebrate central nervous system, glycinergic neurotransmission is regulated by the action of the glycine transporters 1 and 2 (GlyT1 and GlyT2)—members of the solute carrier family 6 (SLC6). Several invertebrate deuterostomes have two paralogous glycine carrier genes, with one gene in the pair having greater sequence identity and higher alignment scores with respect to GlyT1 and the other paralog showing greater similarity to GlyT2. In phylogenetic trees, GlyT2-like sequences from invertebrate deuterostomes form a monophyletic subclade with vertebrate GlyT2, while invertebrate GlyT1-like proteins constitute an outgroup to both the GlyT2-like proteins and to vertebrate GlyT1 sequences. These results are consistent with the hypothesis that vertebrate GlyT1 and GlyT2 are, respectively, derived from GlyT1- and GlyT2-like genes in invertebrate deuterostomes. This implies that the gene duplication which gave rise to these paralogs occurred prior to the origin of vertebrates. GlyT2 subsequently diverged significantly from its invertebrate orthologs (i.e., through the acquisition of a unique N-terminus) as a consequence of functional specialization, being expressed principally in the lower CNS; while GlyT1 has activity in both the lower CNS and several regions of the forebrain.     

Agomelatine and Duloxetine Synergistically Modulates Apoptotic Pathway by Inhibiting Oxidative Stress Triggered Intracellular Calcium Entry in Neuronal PC12 Cells: Role of TRPM2 and Voltage-Gated Calcium Channels

Calcium ion (Ca²⁺) is one of the universal second messengers, which acts in a wide range of cellular processes. Results of recent studies indicated that ROS generated by depression leads to loss of endoplasmic reticulum-Ca²⁺ homeostasis, oxidative stress, and apoptosis. Agomelatine and duloxetine are novel antidepressant and antioxidant drugs and may reduce oxidative stress, apoptosis, and Ca²⁺ entry through TRPM2 and voltage-gated calcium channels. We tested the effects of agomelatine, duloxetine, and their combination on oxidative stress, Ca²⁺ influx, mitochondrial depolarization, apoptosis, and caspase values in the PC-12 neuronal cells. PC-12 neuronal cells were exposed in cell culture and exposed to appropriate non-toxic concentrations and incubation times for agomelatine were determined in the neurons by assessing cell viability. Then PC-12 cells were incubated with agomelatine and duloxetine for 24 h. Treatment of cultured PC-12 cells with agomelatine, duloxetine, and their combination results in a protection on apoptosis, caspase-3, caspase-9, mitochondrial membrane depolarization, cytosolic ROS production, glutathione peroxidase, reduced glutathione, and lipid peroxidation, values. Ca²⁺ entry through non-specific TRPM2 channel blocker (2-APB) and voltage-gated Ca²⁺ channel blockers (verapamil and diltiazem) was modulated by agomelatine and duloxetine. However, effects of duloxetine on the Ca²⁺ entry through TRPM2 channels were higher than in agomelatine. Results of current study suggest that the agomelatine and duloxetine are useful against apoptotic cell death and oxidative stress in PC-12 cells, which seem to be dependent on mitochondrial damage and increased levels of intracellular Ca²⁺ through activation of TRPM2 and voltage-gated Ca²⁺ channels.     

Role of Structural Changes Induced in Biological Membranes by Hydrolysable Tannins from Sumac Leaves (Rhus typhina L.) in their Antihemolytic and Antibacterial Effects

In this study, we found that the sumac tannins (Rhus typhina L.) exert to a various extent antihemolytic effects and antibacterial activity against Bacillus cereus and Pseudomonas aeruginosa depending on structural specificity of bacteria and different mechanisms of their toxic action. The sumac tannins exert the most expressed activity against B. cereus. The antihemolytic effect of the sumac tannins seems to be connected to a greater extent with their modifying action on the erythrocyte membrane structure. It was found that the sumac tannins are incorporated into the erythrocyte membrane, causing transformation of discocytes into echinocytes and enhancing the rigidity of the hydrophilic region of the lipid bilayer. We suggest that the embedding of sumac tannins into the membrane of erythrocytes alters their physical properties and, as a consequence, can limit their interaction with bacterial toxins.     

Chemical composition,antimicrobial, antioxidative and anticholinesterase activity of Satureja Montana L. ssp montana essential oil

The present study investigates the chemical compositions of three Satureja montana L. ssp montana essential oils and correlates chemical variability with biological activities. GC/MS analysis showed that with an increase in altitude (100–500–800 m), a higher content of linalool, terpinen-4-ol and cis-sabinene hydrate was found, while the percentage of phenolic compounds, thymol and carvacrol decreased. Antimicrobial activity of the essential oils was tested against 7 fungal and 23 bacterial strains. The essential oil characterized by the highest content of phenols and alcohols exhibited the highest antimicrobial potential. The correlation analysis showed that the major carriers of the obtained antioxidant activity are oxygenated monoterpenes. All essential oils inhibited human serum cholinesterase activity. High antimicrobial potential, together with moderate antioxidant capacity and strong inhibition of human serum cholinesterase, classifies S. montana essential oil as a natural source of compounds that can be used in the treatment of foodborne and neurological diseases, wound and other infections, as well as for general health improvement.     

Role for the TRPV1 Channel in Insulin Secretion from Pancreatic Beta Cells

Transient receptor potential channels have been put forward as regulators of insulin secretion. A role for the TRPV1 ion channel in insulin secretion has been suggested in pancreatic beta cell lines. We explored whether TRPV1 is functionally expressed in RINm5F and primary beta cells from neonate and adult rats. We examined if capsaicin could activate cationic non-selective currents. Our results show that TRPV1 channels are not functional in insulin-secreting cells, since capsaicin did not produce current activation, not even under culture conditions known to induce the expression of other ion channels in these cells. Although TRPV1 channels seem to be irrelevant for the physiology of isolated beta cells, they may play a role in glucose homeostasis acting through the nerve fibers that regulate islet function. At the physiological level, we observed that Trpv1 ^?/? mice presented lower fasting insulin levels than their wild-type littermates, however, we did not find differences between these experimental groups nor in the glucose tolerance test or in the insulin secretion. However, we did find that the Trpv1 ^?/? mice exhibited a higher insulin sensitivity compared to their wild-type counterparts. Our results demonstrate that TRPV1 does not contribute to glucose-induced insulin secretion in beta cells as was previously thought, but it is possible that it may control insulin sensitivity.     

The Phylogenetic Distribution and Evolution of Enzymes Within the Thymidine Kinase 2-like Gene Family in Metazoa

Deoxyribonucleoside kinases (dNKs) carry out the rate-determining step in the nucleoside salvage pathway within all domains of life where the pathway is present, and, hence, are an indication on whether or not a species/genus retains the ability to salvage deoxyribonucleosides. Here, a phylogenetic tree is constructed for the thymidine kinase 2-like dNK gene family in metazoa. Each enzyme class (deoxycytidine, deoxyguanosine, and deoxythymidine kinases, as well as the multisubstrate dNKs) falls into a monophyletic clade. However, in vertebrates, dCK contains an apparent duplication with one paralog lost in mammals, and a number of crustacean genomes (like Caligus rogercresseyi and Lepeophtheirus salmonis) unexpectedly contain not only the multisubstrate dNKs, related to Drosophila multisubstrate dNK, but also a TK2-like kinase. Additionally, crustaceans (Daphnia, Caligus, and Lepeophtheirus) and some insects (Tribolium, Danaus, Pediculus, and Acyrthosiphon) contain several multisubstrate dNK-like enzymes which group paraphyletically within the arthropod clade. This might suggest that the multisubstrate dNKs underwent multiple rounds of duplications with differential retention of duplicate copies between insect families and more complete retention within some crustaceans and insects. Genomes of several basal animalia contain more than one dNK-like sequence, some of which group outside the remaining eukaryotes (both plants and animals) and/or with bacterial dNKs. Within the vertebrates, the mammalian genomes do not contain the second dCK, while birds, fish, and amphibians do retain it. Phasianidae (chicken and turkey) have lost dGK, while it has been retained in other bird lineages, like zebra finch. Reconstruction of the ancestral sequence between the multisubstrate arthropod dNKs and the TK2 clade of vertebrates followed by homology modeling and discrete molecular dynamics calculations on this sequence were performed to examine the evolutionary path which led to the two different enzyme classes. The structural models showed that the carboxyl terminus of the ancestral sequence is more helical than dNK, in common with TK2, although any implications of this for enzyme specificity will require biochemical validation. Finally, rate-shift and conservation-shift analysis between clades with different specificities uncovered candidate residues outside the active site pocket which may have contributed to differentiation in substrate specificity between enzyme clades.     

Different Incubation Times of Cells After Gene Electrotransfer in Fetal Bovine Serum Affect Cell Viability,but Not Transfection Efficiency

Electroporation as a delivery method is increasingly important in gene therapy, not only in vivo but also in in vitro experimental systems. Different applications of gene electrotransfer require high viability of cells and high transfection efficiency of gene electrotransfer. It was already demonstrated that the addition of fetal bovine serum (FBS) immediately after gene electrotransfer leads to improved cell survival and transfection efficiency. Therefore, the aim of the study was to determine whether prolonged incubation of cells in FBS, for more than standard 5 min, can lead to increased transfection efficiency and improved cell survival. Different murine melanoma and murine and human endothelial cell lines were transfected with plasmid encoding green fluorescent protein and then incubated for different periods of time in FBS (5–30 min). Transfection efficiency was determined by flow cytometry and fluorescence microscopy and cell survival by cell viability assay. Prolonged incubation of cells in FBS after gene electrotransfer had varying effect on cell survival, which was decreased in melanoma cell lines B16F1 and B16F10, minimally affected in SVEC4-10 and HUVEC cells and increased in 2H11 cell at 30 min of incubation time in FBS. On the other hand, transfection efficiency of gene electrotransfer was not affected by long incubation of cell in FBS, regardless of the cell line used. The results of our study emphasize the importance of optimization of gene electrotransfer protocol for particular cells and specific purposes of gene electrotransfer, taking into account the importance of transfection efficiency and cell survival.     

Tonoplast Lipid Composition and Proton Pump of Pineapple Fruit During Low-Temperature Storage and Blackheart Development

Vacuole represents a major storage organelle playing vital roles in pH homoeostasis and cellular detoxification. The chemical and functional properties of tonoplast in response to chilling temperature and their roles in chilling injury are largely unknown. In the current study, lipid composition of tonoplast and the activities of two vacuolar proton pumps, H⁺-ATPase (V-ATPase) and H⁺-pyrophosphatase (V-PPase), were investigated in accordance with the development of blackheart, a form of chilling injury in pineapple fruit (Ananas comosus). Chilling temperature at 10 °C for 1 week induced irreversible blackheart injury in concurrence with a substantial decrease in V-ATPase activity. By contrast, the activity was increased after 1 week at 25 °C. The activity of V-PPase was not changed under both temperatures. Level of total phospholipids of tonoplast decreased at 10 °C, but increased at 25 °C. There was no change at the level of total glycolipids under both temperatures. Thus, low temperature increased the ratio of total glycolipids vs. total phospholipids of tonoplast. Phosphatidylcholine and phosphatidylethanolamine were the predominant phospholipids of tonoplast. Low temperature increased the relative level of phosphatidic acid but decreased the percentage of both phosphatidylcholine and phosphatidylethanolamine. Unsaturated fatty acids accounted for over 60 % of the total tonoplast fatty acids, with C18:1 and C18:2 being predominant. Low temperature significantly decreased the percentage of C18:3. Modification of membrane lipid composition and its effect on the functional property of tonoplast at low temperature were discussed in correlation with their roles in the development of chilling injury in pineapple fruit.     

Phosphoinositide 3-Kinase Pathway Mediates Early Aldosterone Action on Morphology and Epithelial Sodium Channel in Mammalian Renal Epithelia

Involvement of phosphoinositide 3-kinases (PI3Ks) in early aldosterone action on epithelial sodium channel (ENaC) in mammalian renal epithelia was investigated by hopping probe ion conductance microscopy combined with patch-clamping in this study. Aldosterone treatment enlarged the cell volume and elevated the apical membrane of renal mpkCCDc14 epithelia, which resulted in enhancing the open probability of ENaC. Inhibition of PI3K pathway by LY294002 obviously suppressed these aldosterone-induced changes in both cell morphology and ENaC activity. These results indicated the important role of PI3K pathway in early aldosterone action and the close relationship between cell morphology and ENaC activity in mammalian renal epithelia.     

Update on Posaconazole Pharmacokinetics: Comparison of Old and New Formulations

Posaconazole is an extended-spectrum triazole antifungal that until recently was only available as an oral suspension. The absorption of this suspension is limited and is greatly affected by food, gastric pH, and intestinal motility. The systemic availability can be enhanced by administration with a high-fat meal, nutritional supplement, or acidic beverage, and by dividing into multiple daily doses. A new delayed-released tablet dosed once-daily has been developed and was recently approved by the U.S. Food and Drug Administration. This provides an attractive option for healthcare providers and patients because it has more predictable systemic availability and higher serum concentrations. However, the inability to crush or chew this tablet still leaves the need for another form of posaconazole, especially for critically ill patients. Such an option may soon become available, as a new drug application has been submitted to the FDA for a once-daily intravenous formulation of posaconazole.     

Vocal Fold Ion Transport and Mucin Expression Following Acrolein Exposure

The vocal fold epithelium is exposed to inhaled particulates including pollutants during breathing in everyday environments. Yet, our understanding of the effects of pollutants on vocal fold epithelial function is extremely limited. The objective of this study was to investigate the effect of the pollutant acrolein on two vocal fold epithelial mechanisms: ion transport and mucin (MUC) synthesis. These mechanisms were chosen as each plays a critical role in vocal defense and in maintaining surface hydration which is necessary for optimal voice production. Healthy, native porcine vocal folds (N = 85) were excised and exposed to an acrolein or sham challenge. A 60-min acrolein, but not sham challenge significantly reduced ion transport and inhibited cyclic adenosine monophosphate-dependent, increases in ion transport. Decreases in ion transport were associated with reduced sodium absorption. Within the same timeline, no significant acrolein-induced changes in MUC gene or protein expression were observed. These results improve our understanding of the effects of acrolein on key vocal fold epithelial functions and inform the development of future investigations that seek to elucidate the impact of a wide range of pollutant exposures on vocal fold health.     

The Evolution of Reproduction-Related NLRP Genes

NLRP proteins are important components of inflammasomes with a major role in innate immunity. A subset of NLRP genes, with unknown functions, are expressed in oocytes and early embryos. Mutations of Nlrp5 in mice are associated with maternal-effect embryonic lethality and mutations of NLRP7 in women are associated with conception of biparental complete hydatidiform moles (biCHMs), suggesting perturbed processes of genomic imprinting. Recessive mutations on NLRP2/7 in humans are associated with reproductive disorders and appear to be induced by a demethylation of the maternal pronucleus. In this study, we find that radiation of NLRP genes occurred before the common ancestor of Afrotheria and Boreoeutheria, with the clade of oocyte-expressed genes originating before the divergence of marsupial and eutherian mammals. There have been multiple independent duplications of NLRP2 genes one of which produced the NLRP7 gene associated with biCHMs.     

Molecular Evolution and Functional Divergence of the Metallothionein Gene Family in Vertebrates

The metallothionein (MT) gene superfamily consists of metal-binding proteins involved in various metal detoxification and storage mechanisms. The evolution of this gene family in vertebrates has mostly been studied in mammals using sparse taxon or gene sampling. Genomic databases and available data on MT protein function and expression allow a better understanding of the evolution and functional divergence of the different MT types. We recovered 77 MT coding sequences from 20 representative vertebrates with annotated complete genomes. We found multiple MT genes, also in reptiles, which were thought to have only one MT type. Phylogenetic and synteny analyses indicate the existence of a eutherian MT1 and MT2, a tetrapod MT3, an amniote MT4, and fish MT. The optimal gene-tree/species-tree reconciliation analyses identified the best root in the fish clade. Functional analyses reveal variation in hydropathic index among protein domains, likely correlated with their distinct flexibility and metal affinity. Analyses of functional divergence identified amino acid sites correlated with functional divergence among MT types. Uncovering the number of genes and sites possibly correlated with functional divergence will help to design cost-effective MT functional and gene expression studies. This will permit further understanding of the distinct roles and specificity of these proteins and to properly target specific MT for different types of functional studies. Therefore, this work presents a critical background on the molecular evolution and functional divergence of vertebrate MTs to carry out further detailed studies on the relationship between heavy metal metabolism and tolerances among vertebrates.     

Continuum Approaches to Understanding Ion and Peptide Interactions with the Membrane

Experimental and computational studies have shown that cellular membranes deform to stabilize the inclusion of transmembrane (TM) proteins harboring charge. Recent analysis suggests that membrane bending helps to expose charged and polar residues to the aqueous environment and polar head groups. We previously used elasticity theory to identify membrane distortions that minimize the insertion of charged TM peptides into the membrane. Here, we extend our work by showing that it also provides a novel, computationally efficient method for exploring the energetics of ion and small peptide penetration into membranes. First, we show that the continuum method accurately reproduces energy profiles and membrane shapes generated from molecular simulations of bare ion permeation at a fraction of the computational cost. Next, we demonstrate that the dependence of the ion insertion energy on the membrane thickness arises primarily from the elastic properties of the membrane. Moreover, the continuum model readily provides a free energy decomposition into components not easily determined from molecular dynamics. Finally, we show that the energetics of membrane deformation strongly depend on membrane patch size both for ions and peptides. This dependence is particularly strong for peptides based on simulations of a known amphipathic, membrane binding peptide from the human pathogen Toxoplasma gondii. In total, we address shortcomings and advantages that arise from using a variety of computational methods in distinct biological contexts.     

Hormone-like product of vascular tissue stimulating starch accumulation in pith explants of kale and endogenous cytokinins

When stem explants of kale (Brassica oleracea L. var.medullosa), containing pith parenchyma and a strip of vascular tissue, were cultured on simple sucrose medium, a hormone-like factor was transported from the vascular tissue to the adjacent pith, where it stimulated accumulation of starch. Similarly, up to a sevenfold increase of starch content in explants could be induced by cytokinins added to the culture medium. The relative stimulatory effect of several cytokinins (5×10^?6 M) and hormone-like product of vascular tissue (HPVT) in a typical experiment were: control (1.0), trans-zeatin (6.7), HPVT (6.2), N⁶-[2-isopentenyl]adenine (5.4), transzeatin riboside (5.2), N⁶-[2-isopentenyl]adenosine (5.4), kinetin (3.6), 6-benzylaminopurine (3.5), and adenine (2.1). Concentration of endogenous cytokinins was determined using ELISA (trans-zeatin, N⁶-[2-isopentenyl]adenine and their ribosides) andAmaranthus bioassay (total cytokinins). No effect of vascular tissue on the level of endogenous cytokinins in explants was found. The results support the conclusions of previous experiments that the HPVT stimulating starch accumulation is not a cytokinin.