Genetic validation of Aspergillus fumigatus phosphoglucomutase as a viable therapeutic target in invasive aspergillosis

Aspergillus fumigatus is the causative agent of invasive aspergillosis, an infection with mortality rates of up to 50%. The glucan-rich cell wall of A. fumigatus is a protective structure that is absent from human cells and is a potential target for antifungal treatments. Glucan is synthesized from the donor uridine diphosphate glucose, with the conversion of glucose-6-phosphate to glucose-1-phosphate by the enzyme phosphoglucomutase (PGM) representing a key step in its biosynthesis. Here, we explore the possibility of selectively targeting A. fumigatus PGM (AfPGM) as an antifungal treatment strategy. Using a promoter replacement strategy, we constructed a conditional pgm mutant and revealed that pgm is required for A. fumigatus growth and cell wall integrity. In addition, using a fragment screen, we identified the thiol-reactive compound isothiazolone fragment of PGM as targeting a cysteine residue not conserved in the human ortholog. Furthermore, through scaffold exploration, we synthesized a para-aryl derivative (ISFP10) and demonstrated that it inhibits AfPGM with an IC₅₀ of 2 μM and exhibits 50-fold selectivity over the human enzyme. Taken together, our data provide genetic validation of PGM as a therapeutic target and suggest new avenues for inhibiting AfPGM using covalent inhibitors that could serve as tools for chemical validation.     

On the specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein as a probe for nitric oxide

The specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) for nitric oxide was evaluated in in vitro systems. The probe was found fairly specific for nitric oxide. Potential sources of artifacts include the autoxidation of DAF-FM, potentiated by light, and its oxidation by sources of superoxide and peroxyl radicals, leading to fluorescence spectra indistinguishable from those of the nitric oxide adduct. Although DAF-FM reacts with peroxynitrite, this reaction seems to be of secondary importance under quasi-physiological conditions. On the other hand, a simultaneous presence of a nitric oxide source and a superoxide or hydrogen peroxide decreases or increases the fluorescence of DAF-FM, respectively, resulting in biased estimates of nitric oxide production.     

Association of five SNPs with human hair colour in the Polish population

Twenty-two variants (single nucleotide polymorphisms – SNPs) of the genes involved in hair pigmentation (OCA2, HERC2, MC1R, SLC24A5, SLC45A2, TPCN2, TYR, TYRP1) were genotyped in a group of 186 Polish participants, representing a range of hair colours (45 red, 64 blond, 77 dark). A genotype-phenotype association analysis was performed.Using z-statistics we identified three variants highly associated with different hair colour categories (rs12913832:A>G in HERC2, rs1805007:T>C and rs1805008:C>T in MC1R). Two variants: rs1800401:C>T in OCA2 and rs16891982:C>G in SLC45A2 showed a high probability of a relation with hair colour, although that probability did not exceed the threshold of statistical significance after applying the Bonferroni correction. We created and validated mathematical logistic regression models in order to test the usefulness of the sets of polymorphisms for hair colour prediction in the Polish population. We subjected four models to stratified cross-validation. The first model consisted of three polymorphisms that proved to be important in the associative analysis. The second model included, apart from the mentioned polymorphisms, additionally rs16891982:C>G in SLC45A. The third model included, apart from the variants relevant in the associating analysis, rs1800401:C>T in OCA. The fourth model consisted of the set of polymorphisms from the first model supplemented with rs16891982:C>G in SLC45A and rs1800401:C>T in OCA. The validation of our models has shown that the inclusion of rs16891982:C>G in SLC45A and rs1800401:C>T in OCA increases the prediction of red hair in comparison with the algorithm including only rs12913832:A>G in HERC2, rs1805007:T>C and rs1805008:C>T in MC1R. The model consisting of all the five above-mentioned genetic variants has shown good prediction accuracies, expressed by the area under the curve (AUC) of the receiver operating characteristics: 0.84 for the red-haired, 0.82 for the dark-haired and 0.71 for the blond-haired.A genotype-phenotype association analysis brought results similar to those in other studies and confirmed the role of rs16891982:C>G, rs12913832:A>G, rs1805007:T>C and rs1805008:C>T in hair colour determination in the Polish population. Our study demonstrated for the first time the possibility of a share of the rs1800401:C>T SNP in the OCA2 gene in hair colour determination. Including this single nucleotide polymorphism in the actual hair colour predicting models would improve their predictive accuracy.     

Ascorbate Restores Lifespan of Superoxide-dismutase Deficient Yeast

Yeast (Saccharomyces cerevisiae) mutants lacking CuZn-superoxide dismutase (CuZnSOD) are hypersensitive to oxygen and have significantly decreased replicative life span. Both these defects can be ameliorated by exogenous ascorbate. The effect of ascorbate on life span is complicated by auto-oxidation of its compound in the medium. If negative effects of auto-oxidation are prevented by exchange of the medium, ascorbate prolongs not only mean but also maximal replicative life span of the yeast in the atmosphere of air and of pure oxygen. These results demonstrate that life span shortening due to the lack of a vital antioxidant enzyme can be ameliorated by a low-molecular weight antioxidant.     

Effect of proteolysis on the electron spin resonance spectra of maleimide spin labeled erythrocyte membrane

The ratio of low-field amplitudes of weakly and strongly immobilized signals of ESR spectra of a maleimide spin label bound to erythrocyte membranes (hw/hs) increases progressively during incubation at 37 degrees C. This increase is due to the 'self-digestion' of membrane proteins by endogenous proteinases and is attenuated by proteinase inhibitors. Digestion of membranes with chymotrypsin also increases the hw/hs ratio. These results suggest a need for a careful interpretation of data from spin-labeled membrane proteins, especially in experiments involving prolonged incubations of membrane preparations when the proteolytic effects may be significant.     

Proteolytic self-digestion of bovine erythrocyte membranes

"Self-digestion" of bovine erythrocyte membrane proteins was studied in isolated membrane preparations during prolonged incubation at 37 C. Protease activities associated with the membrane result in progressive degradation of all main erythrocyte membrane proteins, in particular spectrin and Band 3, and formation of lower molecular weight products which have been tentatively assigned to parent molecules. Membrane protein "self-digestion" occurs in a broad pH range (2-11), is inhibited by increased ionic strength and by inhibitors of metalloproteases, cysteine and serine proteases, and activated by low concentrations of SDS. "Self-digestion" also takes place in NaOH-stripped erythrocyte membranes. The activity of a protease involved in the "self-digestion", of apparent molecular weight of about 35,000, was renatured after SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins.     

Redox nanoparticles: synthesis,properties and perspectives of use for treatment of neurodegenerative diseases

Oxidative stress (OS) and nitrative stress (NS) accompany many diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Antioxidants have been proposed to counteract OS/NS in these diseases. Nevertheless, the effects of antioxidants are limited and new, more efficient antioxidants are searched for. Redox-active nanoparticles (RNPs), containing antioxidants create a new therapeutical perspective. This review examines the recent literature describing synthesis and potential applications of cerium oxide RNPs, boron cluster-containing and silica containing RNPs, Gd₃N_@C₈₀ encapsulated RNPs, and concentrates on nitroxide-containing RNPs. Nitroxides are promising antioxidants, preventing inter alia glycation and nitration, but their application poses several problems. It can be expected that application of RNPs containing covalently bound nitroxides, showing low toxicity and able to penetrate the blood–brain barrier will be more efficient in the treatment of neurodegenerative disease, in particular AD and PD basing on their effects in cellular and animal models of neurodegenerative diseases.     

Mineral and chemical characteristics,textural parameters,and the mobility of the selected elements of flotation waste,originating from the Polish copper-mining industry

This article discusses the mineralogy and geochemical characteristics of the fresh copper-flotation waste samples. The mobility of As, Cd, Cr, Cu, Ni, Pb, Tl, Zn was investigated by leaching tests. The main mineral phases identified concerned dolomite, quartz, clay minerals, feldspars, and copper-bearing minerals. Chemically, CaO and silica were dominating, along with a significant concentration of precious (Cu), refractory (Cr, Ti, V, Zr), and toxic (As, Cd, Pb) metals. Elements were bound mainly to the residual fraction and sulphides in the following order: Pb > Cu ≈ Tl > As ≈ Zn > Ni ≈ Cr > Cd. The metal mobility patterns expressed as a percentage of total concentrations, were as follows: Cd (42%) > Cr (26%)> Ni (24%) > Zn (23%) > As (22%) > Tl (20%) > Cu (18%) > Pb (2%). Those constituents were released earlier in lower pH values, although Cu, Cr, and Pb were also released in higher alkaline pH values. However, Zn release was not dependent on pH. When L/S values decreased, elements like As, Cr, Cu, Pb, and Tl were released. That process caused decrease of Cd, Ni, and Zn release.