Aspergillus Niger thermostable Cytosine deaminase-dextran conjugates with enhanced structure stability, proteolytic resistance, and Antiproliferative activity.

Cytosine deaminase (CDA) is a prodrug mediating enzyme converting 5-flurocytosine into 5-flurouracil with profound broad-range anticancer activity towards various cell lines. Availability, molecular stability, and catalytic efficiency are the main limiting factors halting the clinical applications of this enzyme on prodrug and gene therapies, thus, screening for CDA with unique biochemical and catalytic properties was the objective. Thermotolerant/ thermophilic fungi could be a distinctive repertoire for enzymes with affordable stability and catalytic efficiency. Among the recovered thermotolerant isolates, Aspergillus niger with optimal growth at 45 °C had the highest CDA productivity. The enzyme was purified, with purification 15.4 folds, molecular mass 48 kDa and 98 kDa, under denaturing and native PAGE, respectively. The purified CDA was covalently conjugated with dextran with the highest immobilization yield of 75%. The free and CDA-dextran conjugates have the same optimum pH 7.4, reaction temperature 37 °C, and pI 4.5, and similar response to the inhibitors and amino acids suicide analogues, ensuring the lack of effect of dextran conjugation on the CDA conformational structure. CDA-Dextran conjugates had more resistance to proteolysis in response to proteinase K and trypsin by 2.9 and 1.5 folds, respectively. CDA-Dextran conjugates displayed a dramatic structural and thermal stability than the free enzyme, authenticating the acquired structural and catalytic stability upon dextran conjugation. The thermal stability of CDA was increased by about 1.5 folds, upon dextran conjugation, as revealed from the half-life time (T1/2). The affinity of CDA-conjugates (Km 0.15 mM) and free CDA (Km 0.22 mM) to deaminate 5-fluorocytosine was increased by 1.5 folds. Upon dextran conjugation, the antiproliferative activity of the CDA towards the different cell lines "MDA-MB, HepG-2, and PC-3" was significantly increased by mediating the prodrug 5-FC. The CDA-dextran conjugates strongly reduce the tumor size and weight of the Ehrlich cells (EAC), dramatically increase the titers of Caspase-independent apoptotic markers PARP-1 and AIF, with no cellular cytotoxic activity, as revealed from the hematological and biochemical parameters.

Versatile Roles of Microbes and Small RNAs in Rice and Planthopper Interactions.

Planthopper infestation in rice causes direct and indirect damage through feeding and viral transmission. Host microbes and small RNAs (sRNAs) play essential roles in regulating biological processes, such as metabolism, development, immunity, and stress responses in eukaryotic organisms, including plants and insects. Recently, advanced metagenomic approaches have facilitated investigations on microbial diversity and its function in insects and plants, highlighting the significance of microbiota in sustaining host life and regulating their interactions with the environment. Recent research has also suggested significant roles for sRNA-regulated genes during rice-planthopper interactions. The response and behavior of the rice plant to planthopper feeding are determined by changes in the host transcriptome, which might be regulated by sRNAs. In addition, the roles of microbial symbionts and sRNAs in the host response to viral infection are complex and involve defense-related changes in the host transcriptomic profile. This review reviews the structure and potential functions of microbes and sRNAs in rice and the associated planthopper species. In addition, the involvement of the microbiota and sRNAs in the rice-planthopper-virus interactions during planthopper infestation and viral infection are discussed.

Microbial cytosine deaminase is a programmable anticancer prodrug mediating enzyme: antibody, and gene directed enzyme prodrug therapy.

Cytosine deaminase (CDA) is a non-mammalian enzyme with powerful activity in mediating the prodrug 5-fluorcytosine (5-FC) into toxic drug 5-fluorouracil (5-FU), as an alternative directed approach for the traditional chemotherapies and radiotherapies of cancer. This enzyme has been frequently reported and characterized from various microorganisms. The therapeutic strategy of 5-FC-CDA involves the administration of CDA followed by the prodrug 5-FC injection to generate cytotoxic 5-FU. The antiproliferative activity of CDA-5-FC elaborates from the higher activity of uracil pathway in tumor cells than normal ones. The main challenge of the therapeutic drug 5-FU are the short half-life, lack of selectivity and emergence of the drug resistance, consistently to the other chemotherapies. So, mediating the 5-FU to the tumor cells by CDA is one of the most feasible approaches to direct the drug to the tumor cells, reducing its toxic effects and improving their pharmacokinetic properties. Nevertheless, the catalytic efficiency, stability, antigenicity and targetability of CDA-5-FC, are the major challenges that limit the clinical application of this approach. Thus, exploring the biochemical properties of CDA from various microorganisms, as well as the approaches for localizing the system of CDA-5-FC to the tumor cells via the antibody directed enzyme prodrug therapy (ADEPT) and gene directed prodrug therapy (GDEPT) were the objectives of this review. Finally, the perspectives for increasing the therapeutic efficacy, and targetability of the CDA-5-FC system were described.

The Importance of Nitric Oxide as the Molecular Basis of the Hydrogen Gas Fumigation-Induced Alleviation of Cd Stress on Ganoderma lucidum.

Whether or not hydrogen gas (H2) can reduce cadmium (Cd) toxicity in Ganoderma lucidum has remained largely unknown. Here, we report that Cd-induced growth inhibition in G. lucidum was significantly alleviated by H2 fumigation or hydrogen-rich water (HRW), evaluated by lower oxidative damage and Cd accumulation. Moreover, the amelioration effects of H2 fumigation were better than of HRW in an optimum concentration of H2 under our experimental conditions. Further results showed that H2-alleviated growth inhibition in G. lucidum was accompanied by increased nitric oxide (NO) level and nitrate reductase (NR) activity under Cd stress. On the other hand, the mitigation effects were reversed after removing endogenous NO with its scavenger cPTIO or inhibiting H2-induced NR activity with sodium tungstate. The role of NO in H2-alleviated growth inhibition under Cd stress was proved to be achieved through a restoration of redox balance, an increase in cysteine and proline contents, and a reduction in Cd accumulation. In summary, these results clearly revealed that NR-dependent NO might be involved in the H2-alleviated Cd toxicity in G. lucidum through rebuilding redox homeostasis, increasing cysteine and proline levels, and reducing Cd accumulation. These findings may open a new window for H2 application in Cd-stressed economically important fungi.