New mycotoxins from the scale insect fungus Aschersonia coffeae Henn. BCC 28712

Nine new mycotoxins; five xanthones 1–5, hydroxanthone 6, and three anthraquinones 7–9, together with nine known compounds; sterigmatocystin (10), demethylsterigmatocystin (11), dihydrodemethylsterigmatocystin (12), sterigmatin (13), austocystin F (14), averufin (15), aflatoxin B1, paeciloquinone A, and zeorin, were isolated from the scale insect fungus Aschersonia coffeae Henn. BCC 28712. The structures of these compounds were elucidated using NMR spectroscopic and MS spectrometric analyses. Compounds 1–3 and 6–9 displayed cytotoxic activity while the xanthone 2 and anthraquinones 8 and 9 also showed antimalarial activity.     

Interactions between cycloguanil derivatives and wild type and resistance-associated mutant Plasmodium falciparum dihydrofolate reductases

Abstract  Comparative molecular field analysis (CoMFA) and quantum chemical calculations were performed on cycloguanil (Cyc) derivatives of the wild type and the quadruple mutant (Asn51Ile, Cys59Arg, Ser108Asn, Ile164Leu) of Plasmodium falciparum dihydrofolate reductase (PfDHFR). The represented CoMFA models of wild type ( and r ² = 0.985) and mutant type ( and r ² = 0.979) can describe the differences of the Cyc structural requirements for the two types of PfDHFR enzymes and can be useful to guide the design of new inhibitors. Moreover, the obtained particular interaction energies between the Cyc and the surrounding residues in the binding pocket indicated that Asn108 of mutant enzyme was the cause of Cyc resistance by producing steric clash with p-Cl of Cyc. Consequently, comparing the energy contributions with the potent flexible WR99210 inhibitor, it was found that the key mutant residue, Asn108, demonstrates attractive interaction with this inhibitor and some residues, Leu46, Ile112, Pro113, Phe116, and Leu119, seem to perform as second binding site with WR99210. Therefore, quantum chemical calculations can be useful for investigating residue interactions to clarify the cause of drug resistance. Graphical Abstract  CoMFA steric contour maps of Cyc derivatives against the quadruple mutant PfDHFR. Electrostatic van der Waals surface interaction of Cyc and the key resistance residue Asn108.      

Assay Development and Identification of the First Plasmodium falciparum 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase Inhibitors

In the fight towards eradication of malaria, identifying compounds active against new drug targets constitutes a key approach. Plasmodium falciparum 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (PfHPPK) has been advanced as a promising target, as being part of the parasite essential folate biosynthesis pathway while having no orthologue in the human genome. However, no drug discovery efforts have been reported on this enzyme. In this study, we conducted a three-step screening of our in-house antifolate library against PfHPPK using a newly designed PfHPPK-GFP protein construct. Combining virtual screening, differential scanning fluorimetry and enzymatic assay, we identified 14 compounds active against PfHPPK. Compounds’ binding modes were investigated by molecular docking, suggesting competitive binding with the HMDP substrate. Cytotoxicity and in vitro ADME properties of hit compounds were also assessed, showing good metabolic stability and low toxicity. The most active compounds displayed low micromolar IC₅₀ against drug-resistant parasites. The reported hit compounds constitute a good starting point for inhibitor development against PfHPPK, as an alternative approach to tackle the malaria parasite.     

Radiation Dosimetry Using Alanine and Electron Paramagnetic Resonance (EPR) Spectroscopy: A New Look at an Old Topic

The detection and quantification by electron paramagnetic resonance (EPR) spectroscopy of stable radicals formed in alanine by exposure to γ-radiation is used as a secondary standard for radiation dosimetry measurements, even though the EPR signal is actually derived from >1 radical with different spectral properties. For high radiation doses, microwave power saturation and spectral linewidths are both dependent on the received dose, and result in non-linear calibration curves. Furthermore, using a high-sensitivity microwave cavity, the power at which EPR signal saturation commences is ~0.3–0.4 mW for samples with irradiation doses ≤10 kGy; these values are an order of magnitude lower than those normally used in alanine dosimetry. In addition, the central peak of the first derivative spectrum, the height of which is commonly used in dosimetry measurements, is the most susceptible to microwave power saturation. Therefore, for high-level dosimetry we now recommend that analyses be performed under non-saturating conditions, and that the spectral acquisition parameters should be determined with a standard irradiated to ≤10 kGy to eliminate any intensity problems associated with variable saturation characteristics. At low radiation doses, variations in spectral saturation characteristics are negligible, and partially saturating conditions along with modulation amplitudes much higher than those normally used can reliably produce improved signal-to-noise ratios and allow extension of the methodology to practical working limits of ~0.1–0.2 Gy.     

DNA hybridization enhancement using piezoelectric microagitation through a liquid coupling medium

In conventional DNA microarray hybridization, delivery of target cDNAs to surface-bounded probes depends solely on diffusion, which is notoriously slow, and thus typically requires 6-20 h to complete. In this study, piezoelectric microagitation through a liquid coupling medium is employed to enhance DNA hybridization efficiency and the results are compared with the standard static hybridization method. DNA hybridization was performed in a sealed aluminium chamber containing DNA microarray glass chip, coupling medium and piezoelectric transducers. 3×SSC (Saline Sodium Citrate) was used as a coupling medium to prevent overheating of the piezoelectric transducers and to effectively transmit ultrasonic wave to the glass chip. Flow visualization using fluidic dye and velocimetry (PTV) technique was applied to observe fluid transport in the hybridization chamber. It was revealed that the dye solution was homogeneously distributed within 10 min under dynamic agitation while it took over 1 h to reach the same level of homogeneity in static condition. Plasmodium falciparum DNA microarrays and total RNA extracted from parasite cells were used as a model for DNA microarray experiments. It was found that the required hybridization time may be substantially reduced from 16 h to 4 h by the use of dynamic hybridization scheme. With the same hybridization time of 16 h, dynamic hybridization resulted in higher fluorescent signals of ～33% and ～24% compared to static hybridization in Cy3 and Cy5 channels, respectively. Additionally, good/effective spots, some of which were not formed by static method, were enhanced and distributed more uniformly over the microarray. Therefore, the developed dynamic hybridization with integrated piezoelectric microagitation platform is highly promising for DNA analysis in molecular biology and medical applications.