Screening method to identify inhibitors of siderophore biosynthesis in the opportunistic fungal pathogen, Aspergillus fumigatus

Aims:  Aspergillus fumigatus is the most common cause of airborne mould infections in immunocompromised patients worldwide. Our aim was to develop a method to identify agents that inhibit siderophore biosynthesis because this pathway is unique to the fungus and is essential for virulence.
Methods and Results:  A high-throughput two-step screening assay was developed using 96-well plates in which fungal growth and siderophore production is assessed spectrophotometrically. If a compound inhibits growth only in iron-limited medium (screen 1), its effect on siderophore production is then determined (screen 2). The proof of concept was demonstrated using a known antifungal agent, amphotericin B, and a strain of A. fumigatus deficient in siderophore production.
Conclusions:  The two-stage screening method clearly identified growth defects in A. fumigatus related specifically to siderophore biosynthesis.
Significance and Impact of the Study:  The increasing incidence of life-threatening fungal infections has produced an urgent need for novel antifungal agents. The method described in this report will facilitate the identification of novel antifungal compounds that inhibit a pathway critical for A. fumigatus virulence and have a reduced probability of affecting host metabolism.     

USP39-mediated deubiquitination of Cyclin B1 promotes tumor cell proliferation and glioma progression

BackgroundThe elevated Cyclin B1 expression contributes to various tumorigenesis and poor prognosis. Cyclin B1 expression could be regulated by ubiquitination and deubiquitination. However, the mechanism of how Cyclin B1 is deubiquitinated and its roles in human glioma remain unclear.MethodsCo-immunoprecipitation and other assays were performed to detect the interacting of Cyclin B1 and USP39. A series of in vitro and in vivo experiments were performed to investigate the effect of USP39 on the tumorigenicity of tumor cells.ResultsUSP39 interacts with Cyclin B1 and stabilizes its expression by deubiquitinating Cyclin B1. Notably, USP39 cleaves the K29-linked polyubiquitin chain on Cyclin B1 at Lys242. Additionally, overexpression of Cyclin B1 rescues the arrested cell cycle at G2/M transition and the suppressed proliferation of glioma cells caused by USP39 knockdown in vitro. Furthermore, USP39 promotes the growth of glioma xenograft in subcutaneous and in situ of nude mice. Finally, in human tumor specimens, the expression levels of USP39 and Cyclin B1 are positively relevant.ConclusionOur data support the evidence that USP39 acts a novel deubiquitinating enzyme of Cyclin B1 and promoted tumor cell proliferation at least in part through Cyclin B1 stabilization, represents a promising therapeutic strategy for tumor patients.     

Application of the direct beta counter Matrix 96 for cytotoxic assays: Simultaneous processing and reading of 96 wells using a51Cr-retention assay

To assess the cytotoxic activity of immune cells, we have developed a⁵¹Cr-retention assay in which the radioactivity retained by⁵¹Cr-labeled target cells, following coincubation with cytotoxic cells, is monitored using the automated Matrix 96 beta counter. The Matrix 96 is designed for simultaneously counting 96 samples isolated from a 96-well microplate. It uses 96 uniform and independent detectors operating on the principle of avalanche gas ionization in the Geiger-Muller mode. Samples must be dry because the detectors are of the open-window type. Therefore, samples from the 96 wells of the microplate are simultaneously harvested onto a filter using the MicroMate 196, a 96-well cell harvester, dried and quantified in the Matrix 96. Usually the⁵¹Cr isotope is measured by the detection of gamma radiation in gamma counters. The Matrix 96, however, monitors Auger electrons, which are also emitted by⁵¹Cr. We have shown that the retention assay can be used to monitor the cytotoxic activity of activated lymphocytes including lymphokine-activated killer cells and tumor-infiltrating lymphocytes against various tumor cell lines. This assay is most suitable for experiments in which low E/T ratios are sufficient to detect highly cytotoxic cells, such as clone screening in cloning assays or in limiting-dilution analysis assays. These assays involve processing and reading large numbers of microplates. In this case, the retention assay monitored in the Matrix 96 will improve the work flow and decrease the amount of radioactive waste.This work was supported by the American Cancer Society grant IN-162-C     

Bacteriorhodopsins containing modified chromophores: A study on the wild type and D96N mutant of Halobacterium salinarum

Modification of the chromophore in bacteriorhodopsin (BR) from ET1001 and D96N strains of Halobacterium salinarum (halobium) was carried out. Purple membranes were decolored by means of light-dependent hydroxylaminolysis. The all-trans -isomers of retinal and its 3,4-didehydro-, 4-keto-, and phenyl analogs were reconstituted into apomembranes. Absorption maxima of the homonymic pigments in both strains were similar. The kinetics of the M-intermediates in the mode of a single turnover of the photocycle induced by a short light flash (532 nm, 15 ns) was compared. For the investigated bacteriorhodopsin analogs the efficiency of the M-intermediate formation did not exhibit any reliable dependence on the point mutation. Both for ET1001 and for D96N strains the M-relaxation of the 4-ketoBR was distinctly biphasic, with the slow phase comprising about 10–15% of the signal amplitude. Replacement of the ionone ring by phenyl caused a weak deceleration of the M relaxation (～1.5-fold decrease in t _1/2). Independence of the photocycle deceleration of the point mutation and chromophore modification was shown for all BR analogs studied.