Polyamine derivatives as inhibitors of trypanothione reductase and assessment of their trypanocidal activities

Trypanothione reductase (TR) occurs exclusively in trypanosomes and leishmania, which are the etiological agents of many diseases. TR plays a vital role in the antioxidant defenses of these parasites and inhibitors of TR have potential as antitrypanosomal agents. We describe the syntheses of several spermine and spermidine derivatives and the inhibiting effects of these compounds on T. cruzi TR. All of the inhibiting compounds displayed competitive inhibition of TR-mediated reduction of trypanothione disulfide. The three most effective compounds studied were N⁴,N⁸-bis(3-phenylpropyl)spermine (12), N⁴,N⁸-bis(2-naphthylmethyl)spermine (14), and N¹,N⁸-bis(2-naphthylmethyl)spermidine (21), with K_i values of 3.5, 5.5 and 9.5 μM, respectively. Compounds 12, 14, and 21 were found to be potent trypanocides in vitro with IC₅₀ values ranging from 0.19 to 0.83 μM against four T. brucei ssp. strains. However, these compounds did not prolong the lives of mice infected with trypanosomes. This work indicates that certain polyamine derivatives which target a unique pathway in Trypanosomatidae have potential as antitrypanosomal agents.     

OSMRβ mutants enhance basal keratinocyte differentiation via inactivation of the STAT5/KLF7 axis in PLCA patients

Dear Editor, Primary localized cutaneous amyloidosis (PLCA) is a skin-limited disorder characterized by deposition of amyloid material in the superficial dermis.According to clinical characteristics,PLCA is divided into lichen,macular,and nodular amyloidosis.PLCA is found worldwide but has a higher incidence in South America and Southeast Asia,such as in Brazil and China (Chang et al.,2014;Tey et al.,2016).     

Loss of Heterozygosity Drives Clonal Diversity of Phytophthora capsici in China

Phytophthora capsici causes significant loss to pepper (Capsicum annum) in China and our goal was to develop single nucleotide polymorphism (SNP) markers for P. capsici and characterize genetic diversity nationwide. Eighteen isolates of P. capsici from locations worldwide were re-sequenced and candidate nuclear and mitochondrial SNPs identified. From 2006 to 2012, 276 isolates of P. capsici were recovered from 136 locations in 27 provinces and genotyped using 45 nuclear and 2 mitochondrial SNPs. There were two main mitochondrial haplotypes and 95 multi-locus genotypes (MLGs) identified. Genetic diversity was geographically structured with a high level of genotypic diversity in the north and on Hainan Island in the south, suggesting outcrossing contributes to diversity in these areas. The remaining areas of China are dominated by four clonal lineages that share mitochondrial haplotypes, are almost exclusively the A1 or A2 mating type and appear to exhibit extensive diversity based on loss of heterozygosity (LOH). Analysis of SNPs directly from infected peppers confirmed LOH in field populations. One clonal lineage is dominant throughout much of the country. The overall implications for long-lived genetically diverse clonal lineages amidst a widely dispersed sexual population are discussed.     

Block of P2X7 receptors could partly reverse the delayed neuronal death in area CA1 of the hippocampus after transient global cerebral ischemia

Transient global ischemia (which closely resembles clinical situations such as cardiac arrest, near drowning or severe systemic hypotension during surgical procedures), often induces delayed neuronal death in the brain, especially in the hippocampal CA1 region. The mechanism of ischemia/reperfusion (I/R) injury is not fully understood. In this study, we have shown that the P2X7 receptor antagonist, BBG, reduced delayed neuronal death in the hippocampal CA1 region after I/R injury; P2X7 receptor expression levels increased before delayed neuronal death after I/R injury; inhibition of the P2X7 receptor reduced I/R-induced microglial microvesicle-like components, IL-1β expression, P38 phosphorylation, and glial activation in hippocampal CA1 region after I/R injury. These results indicate that antagonism of the P2X7 receptor and signaling pathways of microglial MV shedding, such as src-protein tyrosine kinase, P38 MAP kinase and A-SMase, might be a promising therapeutic strategy for clinical treatment of transient global cerebral I/R injury.     

Microbial Electricity Generation Enhances Decabromodiphenyl Ether (BDE-209) Degradation

Due to environmental persistence and biotoxicity of polybrominated diphenyl ethers (PBDEs), it is urgent to develop potential technologies to remediate PBDEs. Introducing electrodes for microbial electricity generation to stimulate the anaerobic degradation of organic pollutants is highly promising for bioremediation. However, it is still not clear whether the degradation of PBDEs could be promoted by this strategy. In this study, we hypothesized that the degradation of PBDEs (e.g., BDE-209) would be enhanced under microbial electricity generation condition. The functional compositions and structures of microbial communities in closed-circuit microbial fuel cell (c-MFC) and open-circuit microbial fuel cell (o-MFC) systems for BDE-209 degradation were detected by a comprehensive functional gene array, GeoChip 4.0, and linked with PBDE degradations. The results indicated that distinctly different microbial community structures were formed between c-MFCs and o-MFCs, and that lower concentrations of BDE-209 and the resulting lower brominated PBDE products were detected in c-MFCs after 70-day performance. The diversity and abundance of a variety of functional genes in c-MFCs were significantly higher than those in o-MFCs. Most genes involved in chlorinated solvent reductive dechlorination, hydroxylation, methoxylation and aromatic hydrocarbon degradation were highly enriched in c-MFCs and significantly positively correlated with the removal of PBDEs. Various other microbial functional genes for carbon, nitrogen, phosphorus and sulfur cycling, as well as energy transformation process, were also significantly increased in c-MFCs. Together, these results suggest that PBDE degradation could be enhanced by introducing the electrodes for microbial electricity generation and by specifically stimulating microbial functional genes.     

Polymerase chain reaction–hybridization method using urease gene sequences for high-throughput Ureaplasma urealyticum and Ureaplasma parvum detection and differentiation

In this article, we discuss the polymerase chain reaction (PCR)–hybridization assay that we developed for high-throughput simultaneous detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum using one set of primers and two specific DNA probes based on urease gene nucleotide sequence differences. First, U. urealyticum and U. parvum DNA samples were specifically amplified using one set of biotin-labeled primers. Furthermore, amine-modified DNA probes, which can specifically react with U. urealyticum or U. parvum DNA, were covalently immobilized to a DNA–BIND plate surface. The plate was then incubated with the PCR products to facilitate sequence-specific DNA binding. Horseradish peroxidase–streptavidin conjugation and a colorimetric assay were used. Based on the results, the PCR–hybridization assay we developed can specifically differentiate U. urealyticum and U. parvum with high sensitivity (95%) compared with cultivation (72.5%). Hence, this study demonstrates a new method for high-throughput simultaneous differentiation and detection of U. urealyticum and U. parvum with high sensitivity. Based on these observations, the PCR–hybridization assay developed in this study is ideal for detecting and discriminating U. urealyticum and U. parvum in clinical applications.     

Strain improvement of <Emphasis Type="Italic">Trichoderma viride</Emphasis> for increased cellulase production by irradiation of electron and <Superscript>12</Superscript>C<Superscript>6+</Superscript>-ion beams

Objectives

To improve cellulase production and activity, Trichoderma viride GSICC 62010 was subjected to mutation involving irradiation with an electron beam and subsequently with a ¹²C⁶⁺-ion beam.

Results

Mutant CIT 626 was the most promising cellulase producer after preliminary and secondary screening. Soluble protein production and cellulase activities were increased mutifold. The optimum temperature, pH and culture time for the maximum cellulase production of the selected mutant were 35 °C, pH 5 and 6 days. The highest cellulase production was obtained using wheat bran. The prepared cellulases from T. viride CIT 626 had twice the hydrolytic performance with sawdust (83 %) than that from the parent strain (42.5 %). Furthermore, molecular studies demonstrated that there were some key mutation sites suggesting that some amino acid changes in the protein caused by base mutations had led to the enhanced cellulase production and activity.

Conclusions

Mutagenesis with electron and ¹²C⁶⁺-ion beams could be developed as an effective tool for improvement of cellulase producing strains.

     

Spontaneous mutations in FgSAD1 suppress the growth defect of the Fgprp4 mutant by affecting tri-snRNP stability and its docking in Fusarium graminearum

FgPrp4, the only kinase in the spliceosome, is not essential for viability, but is important for splicing efficiency in Fusarium graminearum. The Fgprp4 deletion mutant had severe growth defects but often produced spontaneous suppressors with faster growth rate. To better understand the suppression mechanism, we identified and characterized spontaneous mutations in the tri-snRNP-specific protein, FgSad1, which suppressed the growth defects of Fgprp4. The L512P mutation was verified for its suppressive effects on Fgprp4, suggesting that mutations in FgSad1 may have effects involving FgPrp4 phosphorylation on FgSad1. Phosphoproteomics analysis showed that FgSad1 may not be the direct substrate of FgPrp4 kinase. Furthermore, truncation analysis showed that the N-terminal, extra RS-rich region of FgSad1 is critical for its function and is post-translationally modified. The P258S or S269P mutations in FgSad1 increased its interactions with the U5 protein FgPrp8 and the U4/U6 protein FgPrp31, which may result in tri-snRNP stabilization. Additionally, the D76N mutation increased the association of FgSad1 with the U2 snRNP. These data indicate that suppressor mutations in FgSad1 increase the stability of the tri-snRNP and/or the affinity of FgSad1 with U2 snRNP and therefore potentially facilitate the docking of tri-snRNP into the spliceosome.     

The plasma membrane H+-ATPase FgPMA1 regulates the development,pathogenicity, and phenamacril sensitivity of Fusarium graminearum by interacting with FgMyo-5 and FgBmh2

Fusarium graminearum, as the causal agent of Fusarium head blight (FHB), not only causes yield loss, but also contaminates the quality of wheat by producing mycotoxins, such as deoxynivalenol (DON). The plasma membrane H⁺-ATPases play important roles in many growth stages in plants and yeasts, but their functions and regulation in phytopathogenic fungi remain largely unknown. Here we characterized two plasma membrane H⁺-ATPases: FgPMA1 and FgPMA2 in F. graminearum. The FgPMA1 deletion mutant (∆FgPMA1), but not FgPMA2 deletion mutant (∆FgPMA2), was impaired in vegetative growth, pathogenicity, and sexual and asexual development. FgPMA1 was localized to the plasma membrane, and ∆FgPMA1 displayed reduced integrity of plasma membrane. ∆FgPMA1 not only impaired the formation of the toxisome, which is a compartment where DON is produced, but also suppressed the expression level of DON biosynthetic enzymes, decreased DON production, and decreased the amount of mycelial invasion, leading to impaired pathogenicity by exclusively developing disease on inoculation sites of wheat ears and coleoptiles. ∆FgPMA1 exhibited decreased sensitivity to some osmotic stresses, a cell wall-damaging agent (Congo red), a cell membrane-damaging agent (sodium dodecyl sulphate), and heat shock stress. FgMyo-5 is the target of phenamacril used for controlling FHB. We found FgPMA1 interacted with FgMyo-5, and ∆FgPMA1 showed an increased expression level of FgMyo-5, resulting in increased sensitivity to phenamacril, but not to other fungicides. Furthermore, co-immunoprecipitation confirmed that FgPMA1, FgMyo-5, and FgBmh2 (a 14-3-3 protein) form a complex to regulate the sensitivity to phenamacril and biological functions. Collectively, this study identified a novel regulating mechanism of FgPMA1 in pathogenicity and phenamacril sensitivity of F. graminearum.