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.

Effects of norepinephrine infusion during cardiopulmonary bypass on perioperative changes in lactic acid level (Norcal).

INTRODUCTION: Hyperlactatemia, a problem reported in up to 30% of cardiac surgery patients, results from excessive production of or decreased clearance of lactate. It is typically a symptom of tissue hypoperfusion and may be associated with the prevalence of postoperative acute mesenteric ischemia and renal failure, or prolonged intensive care unit (ICU) and hospital stay, and increased 30-day mortality. METHODS AND MEASUREMENTS: Eighty cardiac surgery patients using cardiopulmonary bypass (CPB) were randomly assigned into either a placebo (n = 39) or norepinephrine 0.05-0.2 µg/kg/min (n = 41) as well as norepinephrine boluses during CPB to maintain mean arterial blood pressure (MAP) at 65 to 80 mm Hg. Patient assignments were done after receiving ethical approval to proceed. The primary result was the perioperative changes in lactic acid level. Secondary findings were also recorded, including hemodynamic variables, the incidence of vasoplegia, intraoperative hypotension, myocardial ischemia, the need for vasopressor support, postoperative complications, and mortality. RESULTS: The peak levels and perioperative changes in blood lactate during the first 24 postoperative hours, the number of patients who experienced early hyperlactatemia on admission to the ICU (Placebo: 46.2%, Norepinephrine: 51.2%, p = .650), vasoplegia, hemodynamic changes, incidences of intraoperative hypotension, myocardial ischemia, postoperative complications, and mortality rates were similar in the two groups. Patients in the norepinephrine group received lower intraoperative rescue norepinephrine boluses to maintain the target MAP (p = .039) and had higher MAP values during the CPB and intraoperative blood loss [mean difference [95% confidence interval]; 177 [20.9-334.3] ml, p = .027]. CONCLUSION: norepinephrine and placebo infusions during the CPB with the maintenance of MAP from 65 to 80 mmHg had comparative effects on the changes in blood lactate and incidence of vasoplegia after cardiac surgery. Norepinephrine infusion maintained higher MAP values during the CPB.

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.

Bioprospecting endophytic fungi for bioactive metabolites and use of irradiation to improve their bioactivities.

The search for new bioactive compounds with innovative modes of action and chemistry are desperately needed to tackle the increased emergence of drug-resistant microbes. With this view, this paper was conducted for the isolation, identification, and biological evaluation of fungal endophytes of eleven different plant species. A total of 69 endophytic strains were isolated and tested for the presence of bioactive metabolites with antifungal, antibacterial, anticancer, and antioxidant properties in their extracts. Upon screening, two promising strains were found to have all the before-mentioned activities. These strains were Aspergillus sydowii isolated from the bark of Ricinus communis and Aspergillus flavus isolated from the twigs of Psidium guajava. Major compounds present in extracts of the two strains were identified by GC-Mass analyses. Several well-known bioactive compounds as well as unreported ones were identified in the fungal extracts of the two strains. Furthermore, gamma irradiation (at 1000 Gy) of the fungal cultures resulted in improved bioactivities of extracts from the two strains. These findings recommend the two fungal strains as sources of antimicrobial, anticancer, and antioxidant compounds which may aid in the development of novel drugs. The presented research also explains the high-value of fungal endophytes as untapped sources of bioactive metabolites.