Proteomics and metabolomics analyses of camptothecin-producing Aspergillus terreus reveal the integration of PH domain-containing proteins and peptidylprolyl cis/trans isomerase in restoring the camptothecin biosynthesis.

IMPORTANCE: Decreasing the camptothecin productivity by fungi with storage and subculturing is the challenge that halts their further implementation to be an industrial platform for camptothecin (CPT) production. The highest differentially abundant proteins were Pleckstrin homology (PH) domain-containing proteins and Peptidyl-prolyl cis/trans isomerase that fluctuated with the subculturing of A. terreus with a remarkable relation to CPT biosynthesis and restored with addition of F. elastica microbiome.

Synergistic Plant-Microbe Interactions between Endophytic Actinobacteria and Their Role in Plant Growth Promotion and Biological Control of Cotton under Salt Stress.

Bacterial endophytes are well-acknowledged inoculants to promote plant growth and enhance their resistance toward various pathogens and environmental stresses. In the present study, 71 endophytic strains associated with the medicinal plant Thymus roseus were screened for their plant growth promotion (PGP), and the applicability of potent strains as bioinoculant has been evaluated. Regarding PGP traits, the percentage of strains were positive for the siderophore production (84%), auxin synthesis (69%), diazotrophs (76%), phosphate solubilization (79%), and production of lytic enzymes (i.e., cellulase (64%), lipase (62%), protease (61%), chitinase (34%), and displayed antagonistic activity against Verticillium dahliae (74%) in vitro. The inoculation of strain XIEG05 and XIEG12 enhanced plant tolerance to salt stress significantly (p < 0.05) through the promotion of shoot, root development, and reduced the activities of antioxidant enzymes (SOD, POD, and CAT), compared with uninoculated controls in vivo. Furthermore, inoculation of strain XIEG57 was capable of reducing cotton disease incidence (DI) symptoms caused by V. dahliae at all tested salt concentrations. The GC-MS analysis showed that many compounds are known to have antimicrobial and antifungal activity. Our findings provide valuable information for applying strains XIEG05 and XIEG12 as bioinoculant fertilizers and biological control agent of cotton under saline soil conditions.

Lidocaine vs. tramadol vs. placebo wound infiltration for post-cesarean section pain relief: a randomized controlled trial.

OBJECTIVES: In low-income settings, postoperative pain relief could be challenging as a high patient/nurse ratio limits pain assessment and adequate analgesics administration. The multi-center prospective double-blinded parallel randomized controlled trial was done to compare lidocaine, tramadol, and placebo (saline) intraoperative wound infiltration to relieve post-cesarean section wound pain during the first 24 h. METHODS: Ninety-nine cases were equally randomized into three groups, each containing 33 pregnant women undergoing cesarean section under general anesthesia. During operation, the wound was infiltrated subcutaneously with 20 mL of 2% lidocaine solution in the first group, 2 mg/kg tramadol in the second group, and saline in the third group. The primary outcome was to assess the postoperative pain at 2, 4, 6, 12, and 24 h by the Yes-No-Don't Know (YNDK) Scale, while the secondary outcome was to assess the need for further postoperative analgesia. RESULTS: Wound infiltration with lidocaine or tramadol was effective in pain relief, and both were superior to placebo. Wound infiltration with tramadol was superior to lidocaine in pain relief at 2 h and up to 24 h. CONCLUSIONS: Wound infiltration with tramadol has a more prolonged pain relief effect than lidocaine in post-cesarean section pain relief in patients performing cesarean section under general anesthesia lasting up to 24 h, and both are superior to placebo in pain relief.

Bioprocesses optimization and anticancer activity of camptothecin from Aspergillus flavus, an endophyte of in vitro cultured Astragalus fruticosus.

BACKGROUND: Emerging of endophytic fungi as potent camptothecin producers raise the hope for its commercial production, due to their rapid growth and feasibility of metabolic engineering, nevertheless, their loss of productivity with the fungal storage and subculturing is the challenge. Thus, screening for unique fungal isolate with sustainable camptothecin productivity is the objective of this work. RESULTS: The camptothecin productivity of the fungal endophytes of wild and in vitro cultured Astragalus fruticosus was evaluated. Aspergillus flavus ER, endophyte of A. fruticosus explant, was the potent producer (51.7 µg/l), the chemical identity of putative compound was resolved by UV, HPLC and LC-MS/MS analyses. The purified A. flavus camptothecin displayed a significant activity against HEPG-2 (IC50 0.9 mM), MCF7 and HCT29 (IC50 1.2-1.35 mM). The productivity of camptothecin by A. flavus was increased by 1.6 fold with methyljasmonate. Upon Plackett-Burman Design optimization, the yield of camptothecin was enhanced by 3 fold (150 µg/l) comparing to control. The camptothecin biosynthetic machinery of A. flavus was noticed to be attenuated with subculturing, nevertheless, this biosynthetic potency was restored upon addition of A. fruticosus methanolic extract (1%), ensuring the incidence of specific signals from plant tissues that triggers the expression of camptothecin encoding genes. CONCLUSION: This is the first study deciphering the feasibility of A. flavus for sustainable production of camptothecin upon addition of A. fruticosus extracts, that could be a new platform for camptothecin scaling-up.

Effect of Intra Uterine Granulocyte Colony Stimulating Factor vs. Human Chorionic Gonadotropin at Ovum Pick Up Day on Pregnancy Rate in IVF/ICSI Cases With Recurrent Implantation Failure.

OBJECTIVE: Recurrent implantation failure is defined as failure to achieve clinical pregnancy after the transfer of four or more good-quality embryos in a minimum of three fresh or frozen cycles in a woman aged less than 40 years. The objective is to compare between the effect of intrauterine G-CSF, hCG, and saline solution injection (as placebo) at the day of ovum pick-up on clinical pregnancy, chemical pregnancy, implantation, and miscarriage rates in patients with recurrent implantation failure undergoing IVF/ICSI. METHODS: This prospective, double blind, parallel, randomized controlled trial included 150 patients equally divided into 3 groups, each containing 50 individuals. Subjects in Group 1 received intrauterine injections of G-CSF; Group 2: received intrauterine injections of 500 IU of hCG; and Group 3 received intrauterine injections of saline solution as placebo. The primary outcome measure is clinical pregnancy rate. Secondary outcomes are biochemical pregnancy, implantation, and miscarriage rates. RESULTS: Clinical pregnancy, biochemical pregnancy, and implantation rates were highest in the group given G-CSF and lowest in the group administered saline solution; miscarriage rates were not significantly different between the groups. CONCLUSIONS: Intrauterine administration of G-CSF at a dose of 100 µg/1.0 cc at the time of ovum pick-up is associated with better clinical pregnancy, chemical pregnancy, and implantation rates as compared with intrauterine saline solution administration. Further studies are needed to determine the optimum timing of intrauterine administration of G-CSF that achieves the best results, and longer follow-up is needed to determine take-home baby percentages.

Biosynthesis of polyunsaturated fatty acids by two newly cold-adapted Egyptian marine yeast.

The widespread awareness of polyunsaturated fatty acids (PUFAs) benefits for human health has increased the need for their commercial production. Two oleaginous yeast were isolated from the Mediterranean Sea fish and Red Sea fish Epinephelus aeneus and E. areolatus, respectively. These marine candidates were identified by MALDI-TOF/MS biotyper® as Lodderomyces elongisporus and Rhodotorula mucilaginosa. The effect of incubation temperature (7, 15, and 26 °C) and glucose concentration (3% and 8%) on their lipids content were investigated using Sulfo-Phospho-Vanillin (SPV) assay. Their intercellular lipids were visualized by fluorescence microscope using Nile-Red dye. L. elongisporus and R. mucilaginosa produced 20.04% and 26.79% of Linoleic acid, respectively, on normal Basal-Defatted Medium (BDM). Linoleic acid (21.4-22.7%) and α-Linolenic acid (7.5-10.8%) were produced by R. mucilaginosa and L. elongisporus, on normal BDM at 15 °C. High-Glucose BDM induced a positive effect on the total lipids production that reached its maximum of 48% and 54% by R. mucilaginosa and L. elongisporus, respectively, grown at 15 °C. Remarkably, 12.12% of long-chain 15-Docosenoic acid (C22:1) and 21.49% of Tricosanoic acid (C23:0) were detected in the FAs profile of L. elongisporus, when grown on normal BDM at 26 °C. The present study is the first one reporting the FAs profile of the Egyptian Marine L. elongisporus, and its capability to accumulate high amounts of lipids under appropriate fermentation conditions; thus, it could be considered for scaling up production. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03010-4.

Bioprocess optimization of glutathione production by Saccharomyces boulardii: biochemical characterization of glutathione peroxidase.

The well-known probiotic GRAS Saccharomyces boulardii (CNCM I-745) was used for the first time to produce glutathione (GSH). The culture conditions affecting GSH biosynthesis were screened using a Plackett-Burman design (PBD). Analyzing the regression coefficients for 12 tested variables, yeast extract, glucose, peptone, cysteine, temperature and agitation rate had a positive significant effect on GSH production with a maximum yeild 192 mg/L. The impact of kinetics of adding cysteine was investigated in 19 experiments during the growth time course (0-36 h), and the maximum yield of glutathione (235 mg/L) was obtained by addition of cysteine after 8 h post-inoculation. The most significant variables were further explored at five levels using central composite rotatable design (CCRD), giving a maximum production of GSH (552 mg/L). Using baffled flasks, the yield of GSH was increased to 730 mg/L, i.e., 1.32-fold increment. The two rate-limiting genes of GSH biosynthesis "γ-glutamyl cysteine synthetase (GSH1) and GSH-synthetase (GSH2)" were amplified and sequenced to validate the GSH biosynthetic potency of S. boulardii. The sequences of genes showed 99% similarity with GSH1 and GSH2 genes of S. cerevisiae. Glutathione peroxidase was purified and characterized from S. boulardii with molecular mass and subunit structure of 80 kDa and 35 kDa as revealed from native and SDS-PAGE, ensuring its homodimeric identity. The activity of GPx was reduced by 2.5-fold upon demetallization confirming its metalloproteinic identity. The GPx was strongly inhibited by hydroxylamine and DTNB, ensuring the implication of surface lysine and cysteine residues on the enzyme active site domains.

Transverse versus longitudinal blunt extension of the uterine incision during cesarean section in women with a uterine scar of previous cesarean delivery: A randomized controlled trial.

OBJECTIVE: To compare two different blunt extension techniques of the lower segment transverse uterine incision at cesarean delivery in women with a uterine scar of previous cesarean delivery. METHODS: Study design: Prospective single-blinded parallel multi-center randomized controlled trial involving 392 cases equally divided into two groups. Group one had their incision extended transversely, while group two had their incision extended longitudinally. OUTCOME MEASURES: The primary outcome was the unintended extension of the uterine incision, while the secondary outcomes included the need for additional stitches to achieve hemostasis, the drop in hemoglobin level, uterine vessels injury, and the need for blood transfusion. RESULTS: No significant difference between the transverse and longitudinal extension of the uterine incision during cesarean section as regards unintended uterine extension (P = 0.860), uterine vessel injury (P = 0.501), and cases requiring blood transfusion (P = 0.814). Significantly lower drop in hemoglobin level (P ≤ 0.001) and significantly less need for additional stitches (P ≤ 0.001) in cases with the longitudinal extension of uterine incision. CONCLUSION: In women with a uterine scar of previous cesarean delivery, the blunt longitudinal extension of the uterine incision in the lower segment cesarean section didn't differ from the blunt transverse extension as regards unintended uterine extension but is associated with less hemoglobin drop and less need for additional stitches as compared to transverse extension of the incision. Further studies are needed to assess the long-term complications of both techniques.

Bioprocess Optimization for Exopolysaccharides Production by Ganoderma lucidum in Semi-industrial Scale.

BACKGROUND: For many years, Ganoderma was highly considered as biofactory for the production of different types of bioactive metabolites. Of these bioactive compounds, polysaccharides gained much attention based on their high biotherapeutic properties. Therefore, special attention has been paid during the last years for the production of mushrooms bioactive compounds in a closed cultivation system to shorten the cultivation time and increase the product yield. OBJECTIVES: This work focuses on the development of a simple cultivation strategy for exopolysaccharides (EPS) production using Ganoderma lucidum and submerged cultivation system. METHODS: At first, the best medium supporting EPS production was chosen experimentally from the current published data. Second, like many EPS production processes, carbon and nitrogen concentrations were optimized to support the highest production of polysaccharides in the shake flask level. Furthermore, the process was scaled up in 16-L stirred tank bioreactor. RESULTS: The results clearly demonstrated that the best cultivation strategy was cultivation under controlled pH conditions (pH 5.5). Under this condition, the maximal volumetric and specific yield of EPS production were, 5.0 g/L and 0.42 g/g, respectively. CONCLUSION: The current results clearly demonstrate the high potential use of submerged cultivation system as an alternative to conventional solid-state fermentation for EPS production by G. lucidum. Furthermore, the optimization of both carbon and nitrogen sources concentration and scaling up of the process showed a significant increase in both volumetric and specific EPS production.

Response Surface Methodology for Optimization of Genistein Content in Soy Flour and its Effect on the Antioxidant Activity.

Biotransformation of isoflavones glycosides into the aglycone form is essential to attain the maximum bioavailability. The factors affecting deglycosylation of genistin in soy flour using commercial ß-glucosidase enzyme were evaluated. The presence of genistin in soy flour was confirmed by isolation through chromatographic fractionation and identification by spectral method. Two-levels Plackett-Burman design was applied and effective variables for genistein production were determined. Agitation rate, enzyme concentration, and reaction time, owing to their significant positive effect, and pH, owing to its significant negative effect, were further evaluated using Box-Behnken model. Accordingly the optimal combination of the major reaction affecting factors was "enzyme concentration, 1 IU; agitation speed, 250 rpm; reaction time, 5 h and pH 4. The concentration of genistein can be increased by 9.91 folds (from 0.8 mg/g in the non biotransformed soy flour to 7.93 mg/g in the biotransformed one) using the determined optimal combination of major reaction affecting factors. The antioxidant activity of the non biotransformed and biotransformed soy flour extracts was determined by DPPH method. It was found that biotransformation increase the antioxidant activity by two folds. The concentration causing a 50% reduction of DPPH absorbance (EC50) were 10 and 5 mg/mL for the non biotransformed and biotransformed soy flour extracts, respectively.

Toward Enhancing the Enzymatic Activity of a Novel Fungal Polygalacturonase for Food Industry: Optimization and Biochemical Analysis.

BACKGROUND: The review of literature and patents shows that enhancing the polygalacturonase (PG) production and activity are still required to fulfill the increasing demands. METHODS: A dual optimization process, which involved Plackett-Burman design (PBD), with seven factors, and response surface methodology, was applied to optimize the production of extracellular PG enzyme produced by a novel strain of Aspergillus flavus isolated from rotten orange fruit. The fungal PG was purified and biochemically characterized. RESULTS: Three variables (harvesting time, pH and orange pomace concentration), that were verified to be significant by the PBD analysis, were comprehensively optimized via Box-Behnken design. According to this optimization, the highest PG activity (4073 U/mL) was obtained under pH 7 after 48 h using 40 g/L orange pomace as a substrate, with enhancement in PG activity by 51% compared to the first PBD optimization step. The specific activity of the purified PG was 1608 U/mg with polygalacturonic acid and its molecular weight was 55 kDa. The optimum pH was 5 with relative thermal stability (80%) at 50˚C after 30 min. The PG activity improved in the presence of Cu2+ and Ca2+, while Ba2+, Fe2+ and Zn2+ greatly inhibited the enzyme activity. The obvious Km and Vmax values were 0.8 mg/mL and 2000 µmol/min, respectively. CONCLUSION: This study is a starting point for initial research in the field of optimization and characterization of A. flavus PG. The statistical optimization of A. flavus PG and its biochemical characterization clearly revealed that this fungal strain can be a potential producer of PG which has a wide range of industrial applications.

Biotransformation of soy flour isoflavones by Aspergillus niger NRRL 3122 ß-glucosidase enzyme.

ß-glucosidase enzyme produced from Aspergillus niger NRRL 3122 has been partially purified and characterised. Its molecular weight was 180 KDa. The optimal pH and temperature were 3.98 and 55 °C, respectively. It promoted the hydrolysis of soy flour isoflavone glycosides to their aglycone. Two-level Plackett-Burman design was applied and effective variables for genistein production were determined. Reaction time had a significant positive effect, and pH had a significant negative effect. They were further evaluated using Box-Behnken model. Accordingly, the optimal combination of the major reaction affecting factors was reaction time, 5 h and pH, 4. The concentration of genistein increased by 11.73 folds using this optimal combination. The antioxidant activity of the non-biotransformed and biotransformed soy flour extracts was determined by DPPH method. It was found that biotransformation increased the antioxidant activity by four folds.

Semi-industrial Scale Production of a New Yeast with Probiotic Traits, Cryptococcus sp. YMHS, Isolated from the Red Sea.

A new yeast strain with promising probiotic traits was isolated from the Red Sea water samples. The isolate (YMHS) was subjected to genetic characterization and identified as Cryptococcus sp. Nucleotide sequence analysis of the rRNA gene internal transcribed spacer regions showed 95% sequence similarity between the isolate and Cryptococcus albidus. Cryptococcus sp. YMHS exhibited desirable characteristics of probiotic microorganisms; it has tolerance to low pH in simulated gastric juice, resistance to bile salts, hydrophobic characteristics, broad antimicrobial activity, and in vitro ability to degrade cholesterol. The isolate grew well in a semi-defined medium composed of yeast extract, glucose, KH2PO4, (NH4)2SO4, and MgSO4, yielding cell mass of 2.32 and 5.82 g/l in shake flask and in bioreactor cultures, respectively. Fed-batch cultivation, with controlled pH, increased the biomass gradually in culture, reaching 28.5 g/l after 32 h cultivation. Beside the feasible use as a probiotic, the new strain also could be beneficial in the development of functional foods or novel food preservatives. To our knowledge, this is the first report of yeast with probiotic properties isolated from the Red Sea.

Trichosporon jirovecii-mediated synthesis of cadmium sulfide nanoparticles.

Cadmium sulphide is one of the most promising materials for solar cells and of great interest due to its useful applications in photonics and electronics, thus the development of bio-mediated synthesis of cadmium sulphide nanoparticles (CdS NPs) is one of the essential areas in nanoparticles. The present study demonstrates for the first time the eco-friendly biosynthesis of CdS NPs using the yeast Trichosporon jirovecii. The biosynthesis of CdS NPs were confirmed by UV-Vis spectrum and characterized by X-ray diffraction assay and electron microscopy. Scanning and transmission electron microscope analyses shows the formation of spherical CdS NPs with a size range of about 6-15 nm with a mean Cd:S molar ratio of 1.0:0.98. T. jirovecii produced hydrogen sulfide on cysteine containing medium confirmed by positive cysteine-desulfhydrase activity and the colony color turned yellow on 0.1 mM cadmium containing medium. T. jirovecii tolerance to cadmium was increased by the UV treatment and three 0.6 mM cadmium tolerant mutants were generated upon the UV radiation treatment. The overall results indicated that T. jirovecii could tolerate cadmium toxicity by its conversion into CdS NPs on cysteine containing medium using cysteine-desulfhydrase as a defense response mechanism.

Extracellular biosynthesis of anti-Candida silver nanoparticles using Monascus purpureus.

An eco-friendly process for the silver nanoparticles (Ag-NPs) biosynthesis was investigated using the fungus Monascus purpureus as a safe and commercially used microorganism. M. purpureus growth filtrate was used for the reduction of the aqueous silver nitrate into Ag-NPs with almost 100% size range of 1-7 nm, which was considered as one of the smallest microbial biosynthesized Ag-NPs. The biosynthesized Ag-NPs were structurally characterized using UV, FTIR, DLS, TEM, and XRD. The biosynthesized Ag-NPs were stable after 3 months with no alteration in shape or size. M. purpureus showed no nitrate reductase activity, whereas its pigments reducing power was decreased after nanoparticles formation indicating its role in the Ag-NPs biosynthesis. The synthesized Ag-NPs exhibited strong antimicrobial activity against different bacteria and yeasts species. The anti-Candida activity of M. purpureus culture filtrate was enhanced in the presence of Ag-NPs; the maximum increase in microbial inhibition was observed against Candida albicans with 1.73 increased folds of inhibition zones, followed by their activity against C. tropicalis and C. glabrata with 0.919- and 0.694-folds of increase, respectively. The obtained results suggest that the biosynthesized Ag-NPs offers a promising cost-effective, eco-friendly, and an alternative way to the conventional method of synthesis that could have wide applications in medicine.

Kinetic properties and role of bacterial chitin deacetylase in the bioconversion of chitin to chitosan.

Chitin is an extremely insoluble material with very limited industrial use; however it can be deacetylated to soluble chitosan which has a wide range of applications. The enzymatic deacetylation of various chitin samples was investigated using the bacterial chitin deacetylase (CDA), which was partially purified from Alcaligenes sp. ATCC 55938 growth medium and the kinetic parameters of the enzyme were determined. Also, the efficiency of biocatalyst recycling by immobilization technique was examined. CDA activity reached its maximum (0.419 U/ml) after 18 h of bacterial cultivation. When glycol chitin was used as a substrate, the optimum pH of the enzyme was estimated to be 6 after checking a pH range between 3 and 9, while the optimum temperature was found to be 35°C. Addition of acetate (100 mM) in the assay mixture resulted in 50% loss of enzyme activity. The Km value of the enzyme is 1.6 × 10(-4) µM and Vmax is 24.7 µM/min. The average activity of CDA was 0.38 U/ml for both of immobilized and freely suspended cells after 18 h of bacterial growth. Some related patents are also discussed here.

A novel potentiometric biosensor for selective L-cysteine determination using L-cysteine-desulfhydrase producing Trichosporon jirovecii yeast cells coupled with sulfide electrode.

Trichosporon jirovecii yeast cells are used for the first time as a source of L-cysteine desulfhydrase enzyme (EC 4.4.1.1) and incorporated in a biosensor for determining L-cysteine. The cells are grown under cadmium stress conditions to increase the expression level of the enzyme. The intact cells are immobilized on the membrane of a solid-state Ag2S electrode to provide a simple L-cysteine responsive biosensor. Upon immersion of the sensor in L-cysteine containing solutions, L-cysteine undergoes enzymatic hydrolysis into pyruvate, ammonia and sulfide ion. The rate of sulfide ion formation is potentiometrically measured as a function of L-cysteine concentration. Under optimized conditions (phosphate buffer pH 7, temperature 37+/-1 degrees C and actual weight of immobilized yeast cells 100 mg), a linear relationship between L-cysteine concentration and the initial rate of sulfide liberation (dE/dt) is obtained. The sensor response covers the concentration range of 0.2-150 mg L(-1) (1.7-1250 micromol L(-1)) L-cysteine. Validation of the assay method according to the quality control/quality assurance standards (precision, accuracy, between-day variability, within-day reproducibility, range of measurements and lower limit of detection) reveals remarkable performance characteristics of the proposed biosensor. The sensor is satisfactorily utilized for determination of L-cysteine in some pharmaceutical formulations. The lower limit of detection is approximately 1 micromol L(-1) and the accuracy and precision of the method are 97.5% and +/-1.1%, respectively. Structurally similar sulfur containing compounds such as glutathione, cystine, methionine, and D-cysteine do no interfere.