L-asparaginase is an essential enzyme used in cancer treatment, but its production faces challenges like low yield, high cost, and immunogenicity. Recombinant production is a promising method to overcome these limitations. In this study, response surface methodology (RSM) was used to optimize the production of L-asparaginase 1 from Saccharomyces cerevisiae in Escherichia coli K-12 BW25113. The Box-Behnken design (BBD) was utilized for the RSM modeling, and a total of 29 experiments were conducted. These experiments aimed to examine the impact of different factors, including the concentration of isopropyl-b-LD-thiogalactopyranoside (IPTG), the cell density prior to induction, the duration of induction, and the temperature, on the expression level of L-asparaginase 1. The results revealed that while the post-induction temperature, cell density at induction time, and post-induction time all had a significant influence on the response, the post-induction time exhibited the greatest effect. The optimized conditions (induction at cell density 0.8 with 0.7 mM IPTG for 4 h at 30 °C) resulted in a significant amount of L-asparaginase with a titer of 93.52 μg/mL, which was consistent with the model-based prediction. The study concluded that RSM optimization effectively increased the production of L-asparaginase 1 in E. coli, which could have the potential for large-scale fermentation. Further research can explore using other host cells, optimizing the fermentation process, and examining the effect of other variables to increase production.

• Klíčová slova
• Escherichia coli, Saccharomyces cerevisiae, Cancer treatment, L-asparaginase, Recombinant production, Response surface methodology (RSM),
• MeSH
• asparaginasa * genetika   biosyntéza   metabolismus   MeSH
• Escherichia coli K12 genetika   enzymologie   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• fermentace MeSH
• isopropylthiogalaktosid farmakologie   MeSH
• rekombinantní proteiny * genetika   metabolismus   MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• asparaginasa * MeSH
• isopropylthiogalaktosid MeSH
• rekombinantní proteiny * MeSH

Tumor cells often adapt to amino acid deprivation through metabolic rewiring, compensating for the loss with alternative amino acids/substrates. We have described such a scenario in leukemic cells treated with L-asparaginase (ASNase). Clinical effect of ASNase is based on nutrient stress achieved by its dual enzymatic action which leads to depletion of asparagine and glutamine and is accompanied with elevated aspartate and glutamate concentrations in serum of acute lymphoblastic leukemia patients. We showed that in these limited conditions glutamate uptake compensates for the loss of glutamine availability. Extracellular glutamate flux detection confirms its integration into the TCA cycle and its participation in nucleotide and glutathione synthesis. Importantly, it is glutamate-driven de novo synthesis of glutathione which is the essential metabolic pathway necessary for glutamate's pro-survival effect. In vivo findings support this effect by showing that inhibition of glutamate transporters enhances the therapeutic effect of ASNase. In summary, ASNase induces elevated extracellular glutamate levels under nutrient stress, which leads to a rewiring of intracellular glutamate metabolism and has a negative impact on ASNase treatment.

• MeSH
• akutní lymfatická leukemie farmakoterapie   metabolismus   patologie   MeSH
• asparaginasa * farmakologie   metabolismus   MeSH
• citrátový cyklus účinky léků   MeSH
• glutamin metabolismus   MeSH
• glutathion * metabolismus   MeSH
• kyselina glutamová * metabolismus   MeSH
• lidé MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• protinádorové látky farmakologie   MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• asparaginasa * MeSH
• glutamin MeSH
• glutathion * MeSH
• kyselina glutamová * MeSH
• protinádorové látky MeSH

This study profiled the various endophytic fungi isolated from the orchid Cymbidium sp. and their L-asparaginase production and antioxidant potential. The L-asparaginase production was first screened through qualitative plate screening then quantified by the Nesslerization method. The antioxidant potential was quantified via the 2,2-diphenyl-1-picrylhydrazyl assay. A total of 30 endophytic fungi were isolated and all fungal isolates exhibited various degrees of radical scavenging activities (45.28% to 76.4%). Isolate Lasiodiplodia theobromae (C11) had the highest antioxidant capacity, represented by the lowest IC50 value (5.75 mg/mL) and highest ascorbic acid equivalent antioxidant capacity value (12.17 mg/g). Additionally, 16 isolates produced L-asparaginase (53.33%), which includes primarily species of Fusarium proliferatum, Fusarium fujikuroi, Fusarium incarnatum, and Fusarium oxysporum. A new isolate has also been discovered from Cymbidium orchid, Buergenerula spartinae (C28), which showed the highest L-asparaginase activity (1.736 unit/mL). These findings supported the postulation that medicinal species of Orchidaceae such as Cymbidium sp. harbor endophytes that are producers of L-asparaginase and antioxidants with various potential applications.

• Klíčová slova
• Cymbidium, Antioxidant, Bioactive compounds, Fungal endophytes, L-asparaginase, Orchids,
• MeSH
• antioxidancia * metabolismus   MeSH
• asparaginasa * metabolismus   MeSH
• endofyty * izolace a purifikace   metabolismus   enzymologie   klasifikace   MeSH
• Fusarium enzymologie   metabolismus   izolace a purifikace   MeSH
• fylogeneze MeSH
• houby klasifikace   izolace a purifikace   enzymologie   MeSH
• Orchidaceae * mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antioxidancia * MeSH
• asparaginasa * MeSH

PURPOSE: The AIEOP-BFM ALL 2009 protocol included, at the end of the induction phase, a randomized study of patients with high-risk (HR) ALL to investigate if an intensive exposure to pegylated L-asparaginase (PEG-ASNASE, 2,500 IU/sqm once a week × 4) on top of BFM consolidation phase IB allowed us to decrease minimal residual disease (MRD) and improve outcome. PATIENTS AND METHODS: A total of 1,097 patients presented, from June 2010 to February 2017, with one or more of the following HR criteria: KMT2A::AFF1 rearrangement, hypodiploidy, prednisone poor response, poor bone marrow response at day 15 (Flow MRD ≥10%), or no complete remission (CR) at the end of induction. Of them, 809 (85.1%) were randomly assigned to receive (404) or not receive (405) four weekly doses of PEG-ASNASE. RESULTS: By intention to treat (ITT) analysis, there was no significant difference in the proportion of patients with polimerase chain reaction MRD ≥5 × 10-4 at the end of phase IB in the experimental versus control arm (13.9% v 17.0%, P = .25). The 5-year event-free survival (median follow-up 6.3 years) by ITT in the experimental and control arms was 70.4% (2.3) versus 75.0% (2.2; P = .18), and the 5-year overall survival was 81.5% (2.0) versus 84.0% (1.9; P = .25), respectively. The corresponding 5-year cumulative incidence of death in CR was 9.5% (1.5) versus 5.7% (1.2; P = .08), and that of relapse was 17.7% (1.9) versus 17.2% (1.9), respectively (P = .94). Adverse reactions in phase IB occurred in 22.2% and 8.9% of patients in the experimental and control arm, respectively (P < .001). CONCLUSION: Additional PEG-ASNASE in phase IB did not translate into a benefit for decreasing relapse incidence but was associated with higher toxicity. Further improvements with conventional chemotherapy might be difficult in the context of intensive treatment protocols.

• MeSH
• akutní lymfatická leukemie * MeSH
• asparaginasa * MeSH
• kojenec MeSH
• lidé MeSH
• lokální recidiva nádoru farmakoterapie   MeSH
• polyethylenglykoly MeSH
• prednison škodlivé účinky   MeSH
• přežití bez známek nemoci MeSH
• protokoly protinádorové kombinované chemoterapie škodlivé účinky   MeSH
• randomizované kontrolované studie jako téma MeSH
• recidiva MeSH
• výsledek terapie MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• protokol klinické studie MeSH
• Názvy látek
• asparaginasa * MeSH
• polyethylenglykoly MeSH
• prednison MeSH

The influence of light regulation on the growth and enzyme production of three endolichenic fungal isolates, i.e. Pseudopestalotiopsis theae (EF13), Fusarium solani (EF5), and Xylaria venustula (PH22), was determined. The isolates were exposed to blue, red, green, yellow, white fluorescent light (12 h light-12 h dark photoperiod) (test), and 24 h dark (control) conditions. Results revealed that the alternating light-dark conditions resulted in the formation of dark rings in most fungal isolates but was absent in PH22. Red light induced sporulation while yellow light elicited higher biomass in all isolates (0.19 ± 0.01 g, 0.07 ± 0.00 g, and 0.11 ± 0.00 g, for EF13, PH22, and EF5, respectively) as compared to incubation in the dark. Results also showed that blue light induced higher amylase activity in PH22 (15.31 ± 0.45 U/mL) and L-asparaginase activity in all isolates (0.45 ± 0.01 U/mL, 0.55 ± 0.39 U/mL, and 0.38 ± 0.01 U/mL, for EF13, PH22, and EF5, respectively) compared to both control conditions. Green light enhanced the production of xylanase (6.57 ± 0.42 U/mL, 10.64 ± 0.12 U/mL, and 7.55 ± 0.56 U/mL for EF13, PH22, and EF5, respectively) and cellulase (6.49 ± 0.48 U/mL, 9.57 ± 0.25 U/mL, and 7.28 ± 0.63 U/mL, for EF13, PH22, and EF5, respectively). In contrast, red light was the least effective light treatment as production of enzymes was the least, with lower levels of amylase, cellulase, xylanase, and L-asparaginase detected. To conclude, all three endolichenic fungi are light-responsive, with fungal growth regulated with the use of red light and yellow light, and manipulation of enzyme production via blue and green light.

• Klíčová slova
• Amylase, Biomass, Cellulase, Endolichenic fungi, L-asparaginase, Xylanase,
• MeSH
• amylasy MeSH
• asparaginasa * MeSH
• celulasy * MeSH
• endo-1,4-beta-xylanasy MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• amylasy MeSH
• asparaginasa * MeSH
• celulasy * MeSH
• endo-1,4-beta-xylanasy MeSH

[Figure: see text]

• MeSH
• asparaginasa terapeutické užití   MeSH
• biosyntetické dráhy MeSH
• leukemie * farmakoterapie   MeSH
• lidé MeSH
• protinádorové látky * farmakologie   terapeutické užití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• asparaginasa MeSH
• protinádorové látky * MeSH

L-asparaginase (ASNase) is the principal chemotherapeutic agent against different blood cancers. The risks associated with current clinical preparations demand screening for novel ASNases. Accordingly, the study was conducted to shortlist ASNases having clinically safer profiles from a novel niche, namely, microbes in the gut and hemolymph of apparently healthy Scylla serrata. A four-step strategic approach incorporating the essential requirements for clinically safer profiles was followed. The initial step through plate assay showed five (9.61%) potential ASNase producers. The relative prevalence of ASNase producers was higher in hemolymph (13.33%) than gut (4.5%). The positive isolates were identified as Priestia aryabhattai, Priestia megaterium, Bacillus altitudinis, Shewanella decolorationis, and Chryseomicrobium amylolyticum. Quantitative profiles revealed high ASNase production (114.29 to 287.36 U/mL) without any optimization, with an added advantage of the extracellular production. The second step for substrate specificity studies revealed the absence of L-glutaminase and urease activities in ASNases from C. amylolyticum and P. megaterium, the most desirable properties for safe clinical applications. This is the first report of glutaminase and urease-free ASNase from these two bacteria. The third step ensured type II nature of selected ASNases, the targeted form in clinical applications. The fourth step confirmed the activity and stability in human physiological conditions. Altogether, the results revealed two potential ASNases with clinically compatible profiles.

• MeSH
• asparaginasa MeSH
• Bacteria genetika   MeSH
• glutaminasa MeSH
• hemolymfa MeSH
• krabi * MeSH
• lidé MeSH
• protinádorové látky * terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• asparaginasa MeSH
• glutaminasa MeSH
• protinádorové látky * MeSH

Asparaginase-associated pancreatitis (AAP) frequently affects children treated for acute lymphoblastic leukemia (ALL) causing severe acute and persisting complications. Known risk factors such as asparaginase dosing, older age and single nucleotide polymorphisms (SNPs) have insufficient odds ratios to allow personalized asparaginase therapy. In this study, we explored machine learning strategies for prediction of individual AAP risk. We integrated information on age, sex, and SNPs based on Illumina Omni2.5exome-8 arrays of patients with childhood ALL (N=1564, 244 with AAP 1.0 to 17.9 yo) from 10 international ALL consortia into machine learning models including regression, random forest, AdaBoost and artificial neural networks. A model with only age and sex had area under the receiver operating characteristic curve (ROC-AUC) of 0.62. Inclusion of 6 pancreatitis candidate gene SNPs or 4 validated pancreatitis SNPs boosted ROC-AUC somewhat (0.67) while 30 SNPs, identified through our AAP genome-wide association study cohort, boosted performance (0.80). Most predictive features included rs10273639 (PRSS1-PRSS2), rs10436957 (CTRC), rs13228878 (PRSS1/PRSS2), rs1505495 (GALNTL6), rs4655107 (EPHB2) and age (1 to 7 y). Second AAP following asparaginase re-exposure was predicted with ROC-AUC: 0.65. The machine learning models assist individual-level risk assessment of AAP for future prevention trials, and may legitimize asparaginase re-exposure when AAP risk is predicted to be low.

• MeSH
• akutní lymfatická leukemie * farmakoterapie   genetika   MeSH
• asparaginasa * škodlivé účinky   MeSH
• celogenomová asociační studie MeSH
• dítě MeSH
• lidé MeSH
• pankreatitida * chemicky indukované   genetika   MeSH
• protinádorové látky * škodlivé účinky   MeSH
• strojové učení MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• asparaginasa * MeSH
• protinádorové látky * MeSH

Childhood T-cell acute lymphoblastic leukemia (T-ALL) still remains a therapeutic challenge due to relapses which are resistant to further treatment. L-asparaginase (ASNase) is a key therapy component in pediatric T-ALL and lower sensitivity of leukemia cells to this drug negatively influences overall treatment efficacy and outcome. PTEN protein deletion and/or activation of the PI3K/Akt signaling pathway leading to altered cell growth and metabolism are emerging as a common feature in T-ALL. We herein investigated the relationship amongst PTEN deletion, ASNase sensitivity and glucose metabolism in T-ALL cells. First, we found significant differences in the sensitivity to ASNase amongst T-ALL cell lines. While cell lines more sensitive to ASNase were PTEN wild type (WT) and had no detectable level of phosphorylated Akt (P-Akt), cell lines less sensitive to ASNase were PTEN-null with high P-Akt levels. Pharmacological inhibition of Akt in the PTEN-null cells rendered them more sensitive to ASNase and lowered their glycolytic function which then resembled PTEN WT cells. In primary T-ALL cells, although P-Akt level was not dependent exclusively on PTEN expression, their sensitivity to ASNase could also be increased by pharmacological inhibition of Akt. In summary, we highlight a promising therapeutic option for T-ALL patients with aberrant PTEN/PI3K/Akt signaling.

• MeSH
• asparaginasa * farmakologie   terapeutické užití   MeSH
• dítě MeSH
• fosfatidylinositol-3-kinasy * genetika   metabolismus   MeSH
• fosfohydroláza PTEN * genetika   metabolismus   MeSH
• lidé MeSH
• lymfoblastická leukemie-lymfom z prekurzorových T-buněk * farmakoterapie   genetika   metabolismus   MeSH
• protoonkogenní proteiny c-akt metabolismus   MeSH
• signální transdukce MeSH
• T-lymfocyty metabolismus   MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• asparaginasa * MeSH
• fosfohydroláza PTEN * MeSH
• protoonkogenní proteiny c-akt MeSH
• PTEN protein, human MeSH Prohlížeč

BACKGROUND AND OBJECTIVES: Besides allergic reactions, antibodies against polyethylene glycol (PEG) have been associated with reduced PEG-asparaginase (PEG-ASNase) activity. Population pharmacokinetics (popPK) allow for an in-depth investigation of the influence of anti-PEG antibodies on PEG-ASNase pharmacokinetics. METHODS: PEG-ASNase activity (6261 samples) and anti-PEG antibodies (2082/6412 samples prior to/post administration) in 1444 children with acute lymphoblastic leukaemia treated in the AIEOP-BFM ALL 2009 trial were evaluated. Patients received two doses of PEG-ASNase during induction (2500 U/m2, intravenous, biweekly) and a third dose during reinduction treatment. Anti-PEG IgG and IgM measured prior to and post administration were explored for their influence on the initial clearance of PEG-ASNase using a previously established popPK model. Categorical and continuous antibody data, including each isotype individually as well as in combination, were assessed. RESULTS: High pre-existing levels of anti-PEG antibodies increase the initial drug clearance. Analysed separately, both anti-PEG IgGprior and IgMprior were significant covariates; the stronger effect was observed for anti-PEG IgMprior. Hockey stick models best described the data. For anti-PEG IgMprior, each additional log unit above the estimated cut point was related to a 41.4% increase in initial clearance after the first dose in induction. Antibody levels below the cut point were not associated with an effect on clearance. The combination of both isotypes did not provide additional information compared to anti-PEG IgMprior alone. Antibody levels post administration were not associated with an effect on clearance. CONCLUSION: Pre-existing antibodies against PEG-ASNase significantly increased the initial clearance in a subgroup of patients showing high antibody levels. (Trial registration: EU clinical trials register; EudraCT No: 2007-004270-43; first registered 23 October 2009.).

• MeSH
• akutní lymfatická leukemie * farmakoterapie   MeSH
• asparaginasa MeSH
• dítě MeSH
• lidé MeSH
• polyethylenglykoly farmakokinetika   MeSH
• protinádorové látky * MeSH
• Check Tag
• dítě MeSH
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• klinické zkoušky MeSH
• Názvy látek
• asparaginasa MeSH
• pegaspargase MeSH Prohlížeč
• polyethylenglykoly MeSH
• protinádorové látky * MeSH