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.

Bioprospecting endophytic fungi for antifeedants and larvicides and their enhancement by gamma irradiation.

The search and discovery of new natural products with antifeedant and larvicidal potentials to mitigate harmful insects are scientific pressing issues in the modern agriculture. In this paper, the antifeedant and larvicidal potentials of 69 fungal isolates were screened against the Egyptian cotton leafworm Spodoptera littoralis. A total of 17 isolates showed the insecticidal potentials with three promising isolates. These strains were Aspergillus sydowii, Lasiodiplodia theobromae, and Aspergillus flavus isolated from Ricinus communis (bark), Terminalia arjuna (Bark), and Psidium guajava (twigs), respectively. The effect of gamma irradiation on the antifeedant and larvicidal activities of the three strains was investigated. Exposure of the fungal spores to 1000 Gy of gamma rays significantly intensified both the antifeedant and larvicidal potentials. To identify compounds responsible for these activities, extracts of the three strains were fractionated by thin layer chromatography. The nature of the separated compounds namely, Penitrem A, 1, 3, 5, 8- tetramethyl- 4, 6-diethyl- 7- [2- (methoxycarbonyl)ethyl] porphyrin (from A. sydowii), Penitrem A, 2, 7, 12, 17-Tetramethyl-3, 5:8, 10:13, 15:18, 20-tetrakis (2,2-dimethylpropano) porphyrin (from A. flavus), N,N-Diethyl-3-nitrobenzamide, and Diisooctyl-phthalate (from L. theobromae) were studied by GC-MS analysis. These findings recommend endophytic fungi as promising sources of novel natural compounds to mitigate harmful insects.

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.

Prevalence of Helicobacter pylori infection in patients with chronic hepatitis C.

BACKGROUND: Association between Helicobacter pylori (H. pylori) and chronic hepatitis C (CHC) still remains controversial. This work is concerned with assessing the potential role of H. pylori in the progression of hepatitis C virus (HCV)-related chronic liver disease. RESULTS: A total of 449 individuals constituted this study (200 individuals were used to validate the assay while 249 individuals were used to assess the correlation between H. pylori infection and CHC). H. pylori antigen was quantified in serum samples using ELISA. As a consequence, our findings showed that H. pylori positivity was increased significantly (P = 0.021) with liver fibrosis progression as it was found in 44.45% of fibrotic patients and 71.88% of cirrhotic patients. We demonstrated that patients with F4 were accompanied by a significant (P < 0.05) increase in the concentration of H. pylori antigen displaying 16.52-fold and 1.34-fold increase in its level over F0 and F1-F3, respectively. Patients co-infected with H. pylori and HCV are 3.19 times (219%) more likely to experience cirrhosis than those who are mono-infected with HCV. This suggests that the risk for developing F4 was found to increase upon H. pylori co-infection when compared to CHC mono-infected patients. CONCLUSION: The elevated levels of H. pylori-antigen in HCV/H. pylori co-infection suggest increased susceptibility of co-infected patients for promoting hepatic fibrosis progression.

Lipase from Rhizopus oryzae R1: in-depth characterization, immobilization, and evaluation in biodiesel production.

BACKGROUND: Rhizopus species is among the most well-known lipase producers, and its enzyme is suitable for use in many industrial applications. Our research focuses on the production of lipase utilizing waste besides evaluating its applications. RESULTS: An extracellular lipase was partially purified from the culture broth of Rhizopus oryzae R1 isolate to apparent homogeneity using ammonium sulfate precipitation followed by desalting via dialysis. The partially purified enzyme was non-specific lipase and the utmost activity was recorded at pH 6, 40 °C with high stability for 30 min. The constants Km and Vmax, calculated from the Lineweaver-Burk plot, are 0.3 mg/mL and 208.3 U/mL, respectively. Monovalent metal ions such as Na+ (1 and 5 mM) and K+ (5 mM) were promoters of the lipase to enhance its activity with 110, 105.5, and 106.5%, respectively. Chitosan was used as a perfect support for immobilization via both adsorption and cross-linking in which the latter method attained immobilization efficiency of 99.1% and reusability of 12 cycles. The partially purified enzyme proved its ability in forming methyl oleate (biodiesel) through the esterification of oleic acid and transesterification of olive oil. CONCLUSION: The partially purified and immobilized lipase from Rhizopus oryzae R1 approved excellent efficiency, reusability, and a remarkable role in detergents and biodiesel production.

Antimicrobial activity of certain natural-based plant oils against the antibiotic-resistant acne bacteria.

The unceasing emerging of multidrug-resistant bacteria imposes a global foremost human health threat and discovery of new alternative remedies are necessity. The use of plant essential oil in the treatment of many pathogenic bacteria is promising. Acne vulgaris is the most common skin complaint that fears many people about their aesthetic appearance. In this work we investigated the antibacterial activity of some plant oils against acne-inducing bacteria. Three bacterial isolates were identified from Egypt, biochemically and by means of 16s rRNA gene typing, and were designated as Staphylococcus aureus EG-AE1, Staphylococcus epidermidis EG-AE2 and Cutibacterium acnes EG-AE1. Antibiotic susceptibility test showed resistance of the isolates to at least six antibiotics, yet they are still susceptible to the last resort Vancomycin. In vitro investigations of eleven Egyptian plant oils, identified tea tree and rosemary oils to exhibit antibacterial activity against the antibiotic-resistant acne isolates. Inhibition zones of 15 ± 0.5, 21.02 ± 0.73 and 20.85 ± 0.76 mm was detected when tea tree oil applied against the above-mentioned bacteria respectively, while inhibition zones of 12.5 ± 1.5, 15.18 ± 0.38 and 14.77 ± 0.35 mm were detected by rosemary oils. Tea tree and rosemary oils exhibited bacteriostatic and bactericidal activity against all the strains with MICs/MBCs ranging between 39-78 mg/L for tea tree oil and 39-156 mg/L for rosemary oil. All the isolates were killed after 4 and 6 h upon growing with 200 mg/L of tea tree and rosemary oils, respectively. Additionally, gas chromatography mass spectrometry (GC/MS) profiling identified and detected a variable number of antimicrobial compounds in both oils.

Antibacterial effects of antibiotics and cell-free preparations of probiotics against Staphylococcus aureus and Staphylococcus epidermidis associated with conjunctivitis.

Conjunctivitis, caused by bacterial infections, represents health concern and diagnosis of the disease is pivotal for the proper selection of the treatment. The main causes of bacterial conjunctivitis vary in different countries. The current study investigated the common bacterial causes of bacterial conjunctivitis from eye clinics' attendants and evaluated the effectiveness of different therapeutic approaches. Eye swabs from patients, diagnosed with conjunctivitis, were assessed microbiologically and the isolated bacteria were identified using the standard biochemical identification and sequencing of the 16S rRNA gene. Antibiotics' susceptibility of the conjunctivitis-associated bacterial pathogens was evaluated against nineteen broad-spectrum antibiotics. In the meanwhile, cell-free preparations from probiotic Lactobacillus and Bifidobacterium strains were used to evaluate their antagonistic activities. Findings from this study showed that out of 52 specimen, 17 eye swabs from patients with conjunctivitis were bacterial culture-positive. The identity of the bacterial species, using the biochemical identification system, was Staphylococcus aureus (4 isolates) and S. epidermidis (13 isolates). Staphylococcus spp. showed susceptibility to linezolid, vancomycin, novobiocin, and fluoroquinolones (norfloxacin, ofloxacin, ciprofloxacin and levofloxacin). However, isolates from the two Staphylococcus spp. expressed resistance to penicillin G, oxacillin, and cephalexin. As alternatives to antibiotics, the growth of Staphylococcus spp., including isolates with antibiotic resistance, was inhibited by cell-free preparations of the 4 probiotic Lactobacillus and the 2 Bifidobacterium strains. These findings provide evidence that topical antibiotics such as fluoroquinolones are still effective antimicrobial agents against staphylococci associated with conjunctivitis whereas probiotic preparations could be promising for further research to pave the way for their therapeutic applications against ophthalmic diseases.

Comparison of Three Different Concentrations of Levobupivacaine for Epidural Labor Analgesia: Clinical Effect and Pharmacokinetic Profile.

BACKGROUND: The aim is to compare the clinical effect of three different concentrations of levobupivacaine (0.25%, 0.125%, and 0.0625%) on the sensory and motor block characteristics and mode of delivery during epidural labor analgesia. We also studied the pharmacokinetic profile of the three concentrations during labor. MATERIALS AND METHODS: Sixty pregnant females undergoing normal vaginal delivery under epidural analgesia were divided into three groups according to the concentration of levobupivacaine used. All parturients received an epidural bolus dose of 15 ml of the desired concentration followed by a continuous infusion of the same concentration at 10 mL/h, each combined with fentanyl 2 µg/mL. Sensory block was assessed by the visual analog score (VAS), whereas motor block was evaluated by the Bromage score. Assessments were performed every 5 min in the first 20 min after initiation of epidural analgesia and then at 30 min interval. The incidence of instrumental delivery and cesarean section was also recorded. The total plasma concentrations of levobupivacaine were determined before the start of epidural analgesia, 5 and 10 min after starting the infusion, at infusion stop time, and 3-8 h after infusion termination. RESULTS: The VAS was significantly lower with levobupivacaine concentrations of 0.25% and 0.125% than 0.0625%. Motor block in the form of Bromage score 1 was observed in 39% of parturients receiving levobupivacaine 0.25% of which 43% were converted to cesarean delivery. No motor block was observed with the other two concentrations. Levobupivacaine peak plasma concentrations increased with increasing the concentration of the local anesthetic. There was no difference in other pharmacokinetic parameters between the three groups. CONCLUSION: levobupivacaine concentration of 0.125% is superior to other concentrations for epidural labor analgesia as it provides adequate analgesia without motor affection which reflects in a lower incidence of instrumental delivery or cesarean section.