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Due to the high incidence of nosocomial Candida albicans infection, the first-line drugs for C. albicans infection have been heavily used, and the emergence of drug-resistant strains has gradually increased. Thus, a new antifungal drug or therapeutic method is needed. Chitosan, a product of chitin deacetylation, is considered to be potentially therapeutic for fungal infections because of its excellent biocompatibility, biodegradability and low toxicity. The biocidal action of chitosan against C. albicans shows great commercial potential, but the exact mechanisms underlying its antimicrobial activity are unclear. To reveal these mechanisms, mutant library screening was performed. ADA2 gene, which encodes a histone acetylation coactivator in the SAGA complex, was identified. Transmission electronic microscopy images showed that the surface of chitosan-treated ada2 Δ cells was substantially disrupted and displayed an irregular morphology. Interestingly, the cell wall of ada2 Δ cells was significantly thinner than that of wild-type cells, with a thickness similar to that seen in the chitosan-treated wild-type strain. Although ADA2 is required for chitosan tolerance, expression of ADA2 and several Ada2-mediated cell wall-related genes ( ALS2, PGA45 , and ACE2 ) and efflux transporter genes ( MDR1 and CDR1 ) were significantly inhibited by chitosan. Furthermore, GCN5 encoding a SAGA complex catalytic subunit was inhibited by chitosan, and gcn5 Δ cells exhibited phenotypes comparable to those of ada2 Δ cells in response to chitosan and other cell surface-disrupting agents. This study demonstrated that a potential antifungal mechanism of chitosan against C. albicans operates by inhibiting SAGA complex gene expression, which decreases the protection of the cell surface against chitosan.

An epigenetic transition between white cells and opaque cells influences several properties of Candida albicans biology, including cellular morphology, biofilm formation, virulence, and sexual mating. In particular, these two cell types exhibit marked differences in their ability to undergo sex. A previous study identified the transcriptional regulator of pheromone response in both the white and opaque states as Cph1 because deletion of this gene abolished both pheromone-induced cell adhesion in white cells and sexual mating in opaque cells. To further explore how these cell types exhibit distinct biological outputs upon pheromone stimulation, we selected five Cph1-regulated genes with significant expression during the pheromone response in the white state but not the opaque state. These phase-specific pheromone-induced genes are ORF19.1539, ORF19.1725, ORF19.2430, ORF19.2691 and ORF19.5557. Deletion of each gene revealed that orf19.1539Δ, orf19.1725Δ, orf19.2430Δ and orf19.5557Δ showed significant decreases in pheromone-stimulated cell adhesion in the white state but retained normal mating competency in the opaque state, indicating that a particular role in white cell pheromone response is mediated by these four genes. Interestingly, the defects of orf19.1725Δ in pheromone-stimulated cell adhesion also abolished conventional biofilms and hyphal growth. Zebrafish egg infection assays further demonstrated that ORF19.1725 is involved in cell adhesion, penetration and virulence. Overall, four Cph1-regulated downstream targets were identified in the regulation of white cell pheromone response. We also clarified the roles of C. albicans ORF19.1725 in cell adhesion, hyphal growth, biofilm formation and virulence.

To achieve insulating materials with a low-dielectric characteristic for high-frequency communication applications, three phosphinated poly(aryl ether)s: P1-act (with acetic moiety), P1-mma (with phenyl methacrylic moiety), and P1-vbe (with vinylbenzyl ether moiety) were modified from a phenol-functionalized phosphinated poly(aryl ether) (P1). P1-act and P1-mma, both with active ester linkages (Ph-O-(C=O)-), were reacted with three commercial epoxy resins (diglycidyl ether of bisphenol A, HP7200, and cresol novolac epoxy) to obtain secondary hydroxyl-free epoxy thermosets. Because of the secondary hydroxyl-free structure, epoxy thermosets cured by P1-act and P1-mma show an 11-15% reduction in dielectric constant than those cured by P1. P1-vbe, with reactive vinylbenzyl ether moieties, was self-cured to a high-performance thermoset with a T g value as high as 302 °C and a dielectric constant as low as 2.64 U . High- T g and low-dielectric thermosets have been developed in this work., Competing Interests: The authors declare no competing financial interest.

To achieve high- T g and low-dielectric epoxy thermosets, four dicyclopentadiene-derived polyarylates (26-P, 26-M, 236-P, and 236-M) were prepared from 2,6-dimethyl (or 2,3,6-trimethyl) phenol-dicyclopentadiene adduct with terephthaloyl (or isophthaloyl) chloride by high-temperature solution polymerization. The resulting polyarylates, exhibiting active ester linkages (Ph-O-(C=O)-) are found to be reactive toward a commercial dicyclopentadiene phenol epoxy (HP7200) in the presence of some lone-pair electron-containing compounds. Five compounds including 4-dimethylaminopyridine (DMAP), imidazole, 2-methylimidazole, triphenylphosphine, and triphenylimidazole have been evaluated as a catalyst for the curing reactions. We found that DMAP, with the smallest p K b among them, is the best catalyst according to differential scanning calorimetry, infrared, and thermal analyses. The thermal and dielectric properties of the polyarylate/HP7200 thermosets are evaluated. We found that they exhibit a high T g characteristic (e.g., T g is 238 °C for DMAP-catalyzed, 236-P/HP7200 thermoset). Furthermore, because of the hydrophobic methyl and cycloaliphatic moieties, and the secondary hydroxyl-free structure, polyarylate/HP7200 thermosets show a relative low-dielectric constant of around 2.75 U. The detailed structure-properties relationship is discussed in this work., Competing Interests: The authors declare no competing financial interest.

Three methacrylate-containing polyimides (Px⁻MMA; x = 1⁻3) were prepared from the esterification of hydroxyl-containing polyimides (Px⁻OH; x = 1⁻3) with methacrylic anhydride. Px⁻MMA exhibits active ester linkages (Ph⁻O⁻C(=O)⁻) that can react with epoxy in the presence of 4-dimethylaminopyridine (DMAP), so Px⁻MMA acted as a curing agent for a dicyclopentadiene-phenol epoxy (HP7200) to prepare epoxy thermosets (Px⁻MMA/HP7200; x = 1⁻3) thermosets. For property comparisons, P1⁻OH/HP7200 thermosets were also prepared. The reaction between active ester and epoxy results in an ester linkage, which is less polar than secondary alcohol resulting from the reaction between phenolic OH and epoxy, so P1⁻MMA/HP7200 are more hydrophobic and exhibit better dielectric properties than P1⁻OH/HP7200. The double bond of methacrylate can cure at higher temperatures, leading to epoxy thermosets with a high- T g and moderate-to-low dielectric properties., Competing Interests: The authors declare no conflict of interest.

Candida albicans is an opportunistic human pathogen capable of causing life-threatening infections in immunocompromised patients. C. albicans has a unique morphological transition between white and opaque phases. These two cells differ in virulence, mating capability, biofilm formation, and host-cell interaction. Previous studies revealed that deletion of the SSK2, PBS2, or HOG1 gene resulted in 100% opaque cell formation and suppressed the mating response. Thr-174 and Tyr-176 of the Hog1 protein are important phosphoacceptors and can be activated in response to stimuli. In this study, we first demonstrated the importance of two conserved phosphorylation sites in white-opaque switching, mating, and pheromone-stimulated cell adhesion. Six Hog1 point-mutated strains were generated, including nonphosphorylated strains (Hog1(T174A), Hog1(Y176F), and Hog1(T174A,Y176F)) and negatively charged phosphorylated strains (Hog1(T174D), Hog1(Y176D), and Hog1(T174D,Y176D)). Point mutation on Thr-174, Tyr-176 or in combination with the Hog1 protein in C. albicans MTL homozygous strains stimulated opaque cell formation at a frequency of 100%. Furthermore, mating projections of point-mutated strains were significantly shorter and their mating efficiencies and pheromone-stimulated cell adhesive numbers were lower than those of the wild-type. By investigating the effects of Hog1 phosphorylation in ssk1Δ and sln1Δ, we also demonstrate that the phosphorylation intensity of Hog1p is directly involved in the white-opaque switching. Taken together, the results of our study demonstrate that dual phosphorylation sites of C. albicans are crucial for white-opaque transition, sexual mating, and pheromone-induced cell adhesion., (© The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Candida albicans can stochastically switch between two phenotypes, white and opaque. Opaque cells are the sexually competent form of C. albicans and therefore undergo efficient polarized growth and mating in the presence of pheromone. In contrast, white cells cannot mate, but are induced - under a specialized set of conditions - to form biofilms in response to pheromone. In this work, we compare the genetic regulation of such "pheromone-stimulated" biofilms with that of "conventional" C. albicans biofilms. In particular, we examined a network of six transcriptional regulators (Bcr1, Brg1, Efg1, Tec1, Ndt80, and Rob1) that mediate conventional biofilm formation for their potential roles in pheromone-stimulated biofilm formation. We show that four of the six transcription factors (Bcr1, Brg1, Rob1, and Tec1) promote formation of both conventional and pheromone-stimulated biofilms, indicating they play general roles in cell cohesion and biofilm development. In addition, we identify the master transcriptional regulator of pheromone-stimulated biofilms as C. albicans Cph1, ortholog of Saccharomyces cerevisiae Ste12. Cph1 regulates mating in C. albicans opaque cells, and here we show that Cph1 is also essential for pheromone-stimulated biofilm formation in white cells. In contrast, Cph1 is dispensable for the formation of conventional biofilms. The regulation of pheromone- stimulated biofilm formation was further investigated by transcriptional profiling and genetic analyses. These studies identified 196 genes that are induced by pheromone signaling during biofilm formation. One of these genes, HGC1, is shown to be required for both conventional and pheromone-stimulated biofilm formation. Taken together, these observations compare and contrast the regulation of conventional and pheromone-stimulated biofilm formation in C. albicans, and demonstrate that Cph1 is required for the latter, but not the former.

The pathogenic capability of the tangerine pathotype of Alternaria alternata relies on the production of host-selective ACT toxin. Inoculation of A. alternata in leaves of the citrus quickly induced rapid lipid peroxidation, accumulation of hydrogen peroxide (H(2)O(2)), and cell death, indicative of host defensive response. We previously demonstrated an essential role of the A. alternata AaAP1 gene, encoding a redox-responsive YAP1-like transcription factor, to contribute to fungal pathogenicity. The AaAP1 null mutant fails to incite necrotic lesions. In this study, we show further that the fungal mutant defective at the AaAP1 locus displayed reduced activities for glutathione-S-transferase, glutathione peroxidase, glutathione reductase, and ligninolytic peroxidase, yet retained normal production of ACT toxin. In contrast to the wild-type progenitor and the genetically reverted strain, the mutant strain was unable to detoxify H(2)O(2) effectively and was killed upon exposure to H(2)O(2). The mutant strain induced lower levels of H(2)O(2) accumulation in citrus leaves, compared to those induced by the wild-type or by the genetically reverted strain. Upon exposure to H(2)O(2), A. alternata apparently changed expression of a wide array of the genes regulated by AaAP1. Thus, the impairment of the AaAP1 null mutants to incite necrotic lesions is apparently a consequence of their inability to alleviate the toxicity of ROS, and circumvention of plant defenses is important for the disease process.

hspA encodes a small heat shock protein (sHSP) in Xanthomonas campestris pv. campestris, the causative agent of black rot in cruciferous plants. In this study, two-dimensional gel electrophoresis, promoter activity assays, and Northern hybridization results revealed that HspA expression was induced by heat shock but not by other stresses, although low-level expression was detectable by reverse transcription-polymerase chain reaction (RT-PCR) under normal culture conditions. An hspA mutant exhibited reduced tolerance to heat, especially in the presence of MgSO4, but no change in pathogenicity. Results of size-exclusion chromatography indicated that purified HspA(his), containing six C-terminal histidine residues, formed two different size classes of oligomeric complexes--410 and 820 kDa. In contrast, HspA(ter), the unmodified protein translated from the original hspA gene, formed only the 820-kDa complex. These results suggest that the C-terminus of HspA is important for oligomerization. Both HspA820(his) and HspA410(his) were able to partially protect luciferase against heat-induced aggregation. Unlike other reported sHSPs that commonly capture denaturing proteins in refoldable states until refolded by adenosine triphosphate-dependent chaperone systems, HspA(his) alone was capable of reactivating heat-inactivated EcoRI. Thus, Xanthomonas campestris pv. campestris HspA has potential application as a protective agent during the storage of proteins.

Heterotrimeric G proteins play a profound role in the recognition and transduction of extracellular signals in eukaryotic cells. We characterized the AaG alpha1 gene, encoding a fungal Class I G alpha subunit of the GTP-binding protein, in Alternaria alternata of citrus. Interruption of AaG alpha1 with a marker gene resulted in fungal transformants producing fewer conidia, becoming hypersensitive to KCl, and displaying elevated lipolytic and pectolytic activities. Expression of a functional copy of AaG alpha1 in a null mutant restored all altered phenotypes to the wild type. The AaG alpha1 mutants, whose conidia germinate normally, caused necrotic lesions on citrus indistinguishable from wild type. Application of cAMP, its inhibitors (atropine and theophylline), or 3-isobutyl-1-methylxanthine (IBMX) decreased conidiation in the fungal strains carrying a functional AaG alpha1. In contrast, conidial formation in the null mutants was restored by dibutyryl-cAMP or by a low concentration of cAMP or theophylline (1 mM). Unlike the oxidative stress-responsive AaAP1 transcription activator or the AaFUS3 mitogen-activated protein kinase (MAPK), AaG alpha1 is not required for cellular resistance to oxidative, osmotic, or chemical stress. AaFUS3 has also been demonstrated to be essential for conidial formation, suggesting a possible interaction between AaG alpha1 and AaFUS3 during conidiation even though expression of AaG alpha1 was not affected by AaFUS3 and vice versa. Inactivation of AaG alpha1 suppressed accumulation of transcripts of the AaAP1 gene and the AaHSK1 gene encoding a histidine kinase. These are novel features that have not been previously characterized to be associated with the GTP-binding protein.

Stenotrophomonas maltophilia (Sm), with most of the isolates being resistant to multidrugs, is an opportunistic bacterium causing nosocomial infections. In this study, a novel virulent Sm phage, Smp14, was characterized. Electron microscopy showed that Smp14 resembled members of Myoviridae and adsorbed to poles of the host cells during infection. It lysed 37 of 87 clinical Sm isolates in spot test, displayed a latent period of ca. 20 min, and had a burst size of ca. 150. Its genome (estimated to be 160 kb by PFGE), containing m4C and two unknown modified bases other than m5C and m6A as identified by HPLC, resisted to digestion with many restriction endonucleases except MseI. These properties indicate that it is a novel Sm phage distinct from the previously reported phiSMA5 which has a genome of 250 kb digestible with various restriction enzymes. Sequencing of a 16 kb region revealed 12 ORFs encoding structural proteins sharing 15-45% identities with the homologues from T4-type phages. SDS-PAGE displayed 20 virion proteins, with the most abundant one being the 39 kDa major capsid protein (gp23), which had the N-terminal 52 amino acids removed. Phylogenetic analysis based on gp23 classified Smp14 into a novel single-membered T4-type subgroup.

It has been suggested that bradykinin (BK) plays an important role in regulating neointimal formation after vascular injury. However, implication of BK in the growth of rat vascular smooth muscle cells (VSMCs) is controversial. Therefore, we examined the mitogenic effect of BK on VSMCs associated with activation of mitogen-activated protein kinase (MAPK). Both [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation were activated by BK in time- and concentration-dependent manners. Pretreatment of these cells with neither pertussis toxin nor cholera toxin attenuated the BK-induced responses. Pretreatment of VSMCs with Hoe 140 (a selective B(2) receptor antagonist), U73122 (an inhibitor of phospholipase C), and BAPTA/AM (an intracellular Ca(2+) chelator) inhibited both [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to BK. BK-induced [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation were inhibited by pretreatment of VSMCs with tyrosine kinase inhibitors (genistein and herbimycin A), protein kinase C (PKC) inhibitors (staurosporine, Go-6976, and Ro-318220), an MAPK kinase inhibitor (PD98059), and a p38 MAPK inhibitor (SB203580). Overexpression of the dominant negative mutants, H-Ras-15A and Raf-N4, suppressed p42/p44 MAPK activation induced by BK and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. From these results, we concluded that the mitogenic effect of BK is mediated through activation of the Ras/Raf/MEK/MAPK pathway similar to that of PDGF-BB. BK-mediated MAPK activation was modulated by Ca(2+), PKC, and tyrosine kinase all of which are associated with cell proliferation in rat cultured VSMCs., (Copyright 2003 National Science Council, ROC and S. Karger AG, Basel)