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B-cell acute lymphoblastic leukemia is frequent in Hispanic adolescents and young adults. Outcomes of implementation of pediatric-inspired regimens in low-and middle-income countries are not well known.In this study we treated 94 adolescents and young adults with a local BFM regimen designed to be affordable with the use of native L-asparaginase and mitoxantrone administered in an outpatient fashion, and the of BCR/ABL and measurable residual disease (MRD) determined by high sensitivity flow cytometry for risk stratification.Induction mortality was 11%; 25% of patients had to abandon treatment or be transferred to another health system. Two-year overall (OS) and event free survival (EFS) were 61.5% and 49.8%, MRD-negative patients had a 24-month OS of 85.6% vs. 69.6% (p = .024) and EFS of 76% vs. 45.5% (p = .004). Patients older than 40 years and those who abandoned treatment had worse EFS. Overall drug costs in our regimen were 52% lower than those of CALGB10403. The treatment of AYAs with ALL with an outpatient focus was implemented successfully at a reduced cost. Genetic risk assessment, treatment abandonment and lack of access to novel therapies remain major barriers for improving outcomes.

Cancer cells exhibit higher proliferation rates than normal cells, and as a consequence, a higher nutritional demand for metabolites such as amino acids. Such cells demonstrate high expression of amino acid transporters and are significantly dependent on the external uptake of amino acids. Moreover, some types of cancer cells exhibit oncogenic mutations that render them auxotrophic to certain amino acids. This metabolic difference between tumor and normal cells has been explored for developing anticancer drugs. Enzymes capable of depleting certain amino acids in the bloodstream can be employed to inhibit the proliferation of cancer cells and promote cell death. Certain microbial enzymes, such as l -asparaginase and l -amino acid oxidases, have been studied for this purpose. In this paper, we discuss the role of l -asparaginase, the only enzyme currently used as a chemotherapeutic agent. We also review the studies on a new potential antineoplastic agent, l -lysine α-oxidase, an enzyme of l -amino acid oxidase family.

Over expression of recombinant proteins triggers a cellular stress response (CSR) that down-regulates numerous genes that have a key role in sustaining expression. Instead of trying to individually up-regulate these genes we hypothesized that a superior strategy would be to modulate the expression of global regulators that control the expression of many such downstream genes. Transcriptomic profiling of post induction cultures expressing recombinant asparaginase in Escherichia coli showed the down-regulation of several critical genes many of which were under the control of the global regulator lrp which is known to have a significant impact on both amino acid metabolism and protein translation. Therefore, to ameliorate the deleterious effects of the CSR we decided to supplement the activity of lrp using plasmid-based co-expression. We observed that the test culture containing an additional plasmid expressing lrp under the arabinose promoter gave a 50% higher yield of recombinant L-Asparaginase after 32 h in batch culture compared to the control, which had only one plasmid expressing the recombinant protein. This approach helped us design a better performing strain, which could sustain expression rates for a significantly longer time period. This work illustrates that modifying the expression of regulatory genes could serve as a better strategy to prevent the reprogramming of the cellular machinery which is the hallmark of the CSR and help in the design better hosts for recombinant protein expression.

PEGylated-l-asparaginase (PEG-ASNase) is a chemotherapeutic agent used to treat pediatric acute lymphoblastic leukemia (ALL). Its use is avoided in adults due to its high risk of liver injury including hepatic steatosis, with obesity and older age considered risk factors of the injury. Our study aims to elucidate the mechanism of PEG-ASNase-induced liver injury. Mice received 1500 U/kg of PEG-ASNase and were sacrificed 1, 3, 5, and 7 days after drug administration. Liver triglycerides were quantified, and plasma bilirubin, ALT, AST, and non-esterified fatty acids (NEFA) were measured. The mRNA and protein levels of genes involved in hepatic fatty acid synthesis, β-oxidation, very low-density lipoprotein (VLDL) secretion, and white adipose tissue (WAT) lipolysis were determined. Mice developed hepatic steatosis after PEG-ASNase, which associated with increases in bilirubin, ALT, and AST. The hepatic genes Ppara, Lcad/Mcad, Hadhb, Apob100, and Mttp were upregulated, and Srebp-1c and Fas were downregulated after PEG-ASNase. Increased plasma NEFA, WAT loss, and adipose tissue lipolysis were also observed after PEG-ASNase. Furthermore, we found that PEG-ASNase-induced liver injury was exacerbated in obese and aged mice, consistent with clinical studies of ASNase-induced liver injury. Our data suggest that PEG-ASNase-induced liver injury is due to drug-induced lipolysis and lipid redistribution to the liver., Graphical abstract PEG-ASNase-induced liver injury was exacerbated in obese and aged mice, consistent with clinical studies of ASNase-induced liver injury. This study suggests that PEG-ASNase-induced liver injury is due to drug-induced lipolysis and lipid redistribution to the liver.Image 1

This review focuses on asparaginase, a key component of childhood acute lymphoblastic leukaemia (ALL) treatment since the 1970s. This review evaluates how much asparaginase is needed for optimal outcome in childhood ALL. We provide an overview of asparaginase dose intensity, i.e. duration of total cumulative exposure in weeks and level of exposure reflected by dose and/or asparaginase activity level, and the corresponding outcome. We systematically searched papers published between January 1990 and March 2021 in the PubMed and MEDLINE databases and included 20 papers. The level and duration of exposure were based on the pharmacokinetic profile of the drug and the assumption that trough asparaginase activity levels of ≥100 IU/L should be achieved for complete l-asparagine depletion. The statistical meta-analysis of outcomes was not performed because different outcome measures were used. The level of exposure was not associated with the outcome as long as therapeutic asparaginase activity levels of ≥100 IU/L were reached. Conflicting results were found in the randomised controlled trials, but all truncation studies showed that the duration of exposure (expressed as weeks of l-asparagine depletion) does affect the outcome; however, no clear cutoff for optimal exposure duration was determined. Optimal exposure duration will also depend on immunophenotype, (cyto)genetic subgroups, risk group stratification and backbone therapy.

The hyper-CVAD/methotrexate-cytarabine (H-CVAD/ MTX-AraC) chemotherapy protocol has been one of the standard treatments for hematological malignancies, such as mantle cell lymphoma (MCL), Burkitt lymphoma (BL), and B-cell and T-cell acute lymphoblastic leukemia. Because results of this therapy are poor, it has been progressively replaced with new specific regimens with better efficacy profiles (GELA protocol for MCL, BURKIMAB for BL, and PETHEMA for B-cell and T-cell acute lymphoblastic leukemia [ALL]). The objective of this study was to analyze the response rates and survival of these therapeutic regimens. This retrospective and descriptive observational study of 81 patients compared 42 patients treated with hyper-CVAD/methotrexate-cytarabine (group A) with 39 patients treated with GELA/BURKIMAB/PETHEMA (group B). More patients in group B than in group A completed the treatment (89.7% vs. 47.6%, p0.001). In group A, 14.3% did not complete treatment because of death compared with 7.7% in group B, and 29% in group A had cycle delays versus 6.7% in group B (p0.001). In group A, 78.6% of group A achieved a complete response (CR) compared with 94.9% of group B (p = 0.050). Disease-free survival (DFS) and overall survival (OS) were significantly higher in group B (p0.001 in both cases). Data for current therapeutic protocols have indicated superior efficacy, with higher complete response rates, as well as better disease-free survival and overall survival results. This article provides the best results in terms of the efficacy of treatment of four hematological malignancies (MCL, BL, B-cell ALL, and T-cell ALL) with the most current specific therapeutic regimens (GELA for MCL, BURKIMAB for BL, and PETHEMA for B-cell ALL and T-cell ALL) with respect to a classic general protocol (H-CVAD/MTX-AraC for all four). These results may represent a great advance in the treatment of these blood cancers, achieving an important long-term benefit for these hematological patients.

Introduction The extension of pediatric-inspired regimens to young adults with newly diagnosed acute lymphoblastic leukemia (ALL) has considerably improved leukemia outcomes in this population,1–3 and nowadays, more young adults are being cured with chemotherapy and successfully spared from complications of early allogeneic hematopoietic cell transplantation consolidation.4 Pediatric-inspired ALL regimens rely heavily on the use of non-myelosuppressive drugs, such as asparaginase, steroid, and vincristine. Asparaginase, in particular, is a key element for pediatric-inspired regimens, and the inclusion of asparaginase and the more extensive use of it in these regimens has led to better ALL outcomes.5,6 In contrast, a summary of approximately 8,000 children enrolled in the Children’s Oncology Group showed a direct correlation between early asparaginase discontinuation as the result of emerging toxicities and inferior outcomes. 7

Herein, our suggestion is to immobilize enzymes in-situ on absorbable shape-memory stents instead of injecting therapeutic enzymes into the blood. Chitosan (CHI)-based stents were tailored as novel support and the enzyme-immobilizing ability was elucidated using L-asparaginase (L-ASNase). For developing shape-memory stents, CHI-glycerol (GLY) solution was prepared and further blended with different ratios of polyethylene glycol (PEG), and polyvinyl alcohol (PVA). Afterward, the blends were modified by ionic crosslinking with sodium tripolyphosphate to obtain a shape-memory character. L-ASNase was included in the blends by using in-situ method before ionic crosslinking. The prepared stents, with or without L-ASNase, were comprehensively characterized by using several techniques. Collectively, immobilized L-ASNase exhibited much better performance in immobilization parameters than free one, thanks to its improved stability and reusability. For instance, CHI/GLY/PEG-3@L-ASNase retained about 70% of the initial activity after storage at 30 °C for 2 weeks, whereas the free form lost half of its initial activity. Besides, it retained 73.4% residual activity after 15 consecutive cycles. Most importantly, stent formulations exhibited ~60% activity in the bioreactor system after 4 weeks of incubation. Given the above results, shape-memory stents can be a promising candidate as a new platform for immobilization, especially in the blood circulation system.

Clinical Practice Points • Aggressive NK-cell leukemia/lymphoma (ANKL) is a rare natural killer (NK)-cell malignancy, and although it typically presents as rapidly progressive disease, cases may present more indolently, progressing over months rather than weeks. • Unlike the more common extranodal NK-cell leukemia/lymphoma, ANKL may not involve the nasopharynx and can be Epstein-Barr virus (EBV) negative. • Diagnosing ANKL can be challenging. Although not yet standard of care, next-generation sequencing and digital image analysis can be used to clarify this rare diagnosis and confirm EBV negativity. • Treatment of ANKL utilizes asparaginase-based regimens, unlike the anthracycline-based regimens used in peripheral T-cell lymphomas or myeloid disorders. Ensuring the correct diagnosis is crucial in determining the appropriate treatment regimen for this uncommon disease.

L-asparaginase (EC 3.5.1.1) showed great commercial value owing to its effective treatment of acute lymphoblastic leukemia (ALL), lymphoid system malignancies and Hodgkin disease, and also to its use in the prevention of acrylamide formation in fried and baked foods. In this study, a type I L-asparaginase gene from Bacillus licheniformis Z-1 (BlAase) was cloned and expressed in Bacillus subtilis RIK 1285. Results showed that even without the mediation of any N-terminal signal peptides, BlAase can efficiently secrete into the medium. Further investigation indicated that the secretion of the BlAase was via neither Sec- nor Tat-dependent secretion pathway, and both the N- and C-terminal regions of the BlAase were essential for its expression and secretion, implying that BlAase might be secreted via a non-classical secretion pathway. To explore its secretion ability, BlAase was used as a signal peptide to direct the secretion of various heterologous proteins, where two of five proteins were successfully secreted with the mediation of BlAase. To the best of our knowledge, this is the first time to achieve extracellular expression of L-asparaginase via non-classical protein secretion pathway in B. subtilis, and provide a potential tool for secretion of recombinant proteins expressed in B. subtilis using BlAase as a signal peptide.

L-asparaginase is an enzyme capable of hydrolyzing the asparagine to aspartic acid and ammonia. L-asparaginase is widely used in the treatment of acute lymphoblastic leukemia (ALL) and other cancers. Here, for the first time, the effects of a novel yeast L-asparaginase from Yarrowia lipolytica were studied on human lung (A549) and breast cancer (MCF7) cell lines as the solid cancer cell lines in terms of cell growth and metastasis inhibition. Functional analysis showed the L-asparagine deprivation mediated anti-proliferation effects by apoptosis induction and changes in the expression of target genes involved in apoptosis and migration pathways. The qRT-PCR analysis showed the higher expression levels of pro-apoptosis genes, including Bax, P53, caspase 3, caspase 8, and down-regulation of Bcl-2 anti-apoptotic gene in treated cells. On the other hand, there was no increase in ROS production in the treated cells. However, L-asparaginase treatment was able to significantly induce autophagy activation in A549 cells. Besides, wound healing assay showed that L-asparaginase could considerably inhibit the migration of A549 and MCF7 cells. Taken together, our results suggested that Yarrowia lipolytica L-asparaginase might be considered for enzyme therapy against breast and lung cancers. L-asparaginase is an enzyme capable of hydrolyzing the asparagine to aspartic acid and ammonia. L-asparaginase is widely used in the treatment of acute lymphoblastic leukemia (ALL) and other cancers. Here, for the first time, the effects of a novel yeast L-asparaginase from Yarrowia lipolytica were studied on human lung (A549) and breast cancer (MCF7) cell lines as the solid cancer cell lines in terms of cell growth and metastasis inhibition. Functional analysis showed the L-asparagine deprivation mediated anti-proliferation effects by apoptosis induction and changes in the expression of target genes involved in apoptosis and migration pathways. The qRT-PCR analysis showed the higher expression levels of pro-apoptosis genes, including Bax, P53, caspase 3, caspase 8, and down-regulation of Bcl-2 anti-apoptotic gene in treated cells. On the other hand, there was no increase in ROS production in the treated cells. However, L-asparaginase treatment was able to significantly induce autophagy activation in A549 cells. Besides, wound healing assay showed that L-asparaginase could considerably inhibit the migration of A549 and MCF7 cells. Taken together, our results suggested that Yarrowia lipolytica L-asparaginase might be considered for enzyme therapy against breast and lung cancers.

For two decades, the prognostic of adult patients with ALL was improved based on pediatric-inspired protocols. These approaches based on less myelosuppressive drugs have led to improved response rates, decreased relapse rates, with a benefit in survival observed in patients aged up to 50-60-years-old. Therapeutic intensification came with a decrease in the use of allogeneic hematopoietic stem cell transplantation, with current indications mainly based on the level of measurable residual disease. Pediatric approaches are however limited in older patients or in patients with comorbidities, who are at greater risk to develop adverse effects especially to asparaginase. Future progresses will arise from personalized medicine including targeted therapy in some ALL oncogenic subgroups and immunotherapy. Monoclonal antibodies, bispecific antibodies, antibody drug conjugates and CAR-T cells have shown encouraging results in relapsed/refractory diseases. These strategies are now evaluated frontline in children and adults to further increase the quality of response, to limit the toxicity of treatments including allogeneic transplant. The objective of this review is to discuss the benefit and the limits of pediatric therapeutic strategies in adults and the perspectives offered by new approaches including immunotherapies.

Background Currently, pediatric-inspired regimens are commonly applied to adults with acute lymphoblastic leukemia (ALL) after the recent recognition that these protocols improve survival. While asparaginase in whatever available formulation is a key component of modern treatment of ALL, many adult oncologists and hematologists struggle to deal with its particular toxicities in clinical practice. We reviewed toxicity outcomes of pegylated asparaginase (PEG-ASP) in adults with ALL treated in 3 reference centers in Brazil. Patients and Methods This was a cross-sectional retrospective chart-review study encompassing patients aged 15 years and older diagnosed with ALL or ambiguous-lineage leukemia who received at least one dose of PEG-ASP, regardless of the adopted regimen. Results A total of 57 patients were included (age range, 15-57 years). Most patients (70%) received 2000 IU/m2 as the initial dose, by intravenous route (72%). The incidence of thromboembolic events was 17.5%, and the main site was cerebral venous sinus (4/10). Thrombosis was more frequent in patients receiving second-line treatment. In obese patients, grade 3 hepatotoxicity and hyperbilirubinemia were more common. Clinical pancreatitis (grade 3 or higher) was found in 2 of 57 cases. PEG-ASP had to be discontinued in 19.3% of exposed patients (11/57). Conclusion By reviewing the medical charts of adult patients with ALL from 3 reference centers, we found that our incidence of thrombotic and hepatic adverse events is similar to those reported in other trials involving PEG-ASP. Usually these effects should not preclude further use of the drug because most events are manageable in routine clinical practice.

L-Asparaginase (L-ASNase EC 3.5.1.1) is considered as an important biopharmaceutical drug enzyme in the treatment of childhood acute lymphoblastic leukemia (ALL). In the present study, Pyrococcus furiosus L-ASNase gene was cloned into pET26b (+), expressed in E. coli BL21(DE3) pLysS, and purified to homogeneity using Ni2+ chelated Fast Flow Sepharose resin with 5.7 purification fold and 23.9% recovery. The purified enzyme exhibited a molecular weight of ~33,660 Da on SDS-PAGE and showed maximal activity at 50 °C and pH 8.0. It retained 98.3% and 60.7% initial activity after 60 min at 37 °C and 50 °C, respectively. The recombinant enzyme showed highest substrate specificity towards L-ASNase substrate, while no detectable specificity was observed for l -glutamine, urea, and acrylamide at 10 mM concentration. The Km and Vmax of the purified recombinant enzyme as calculated using Lineweaver-Burk plot were determined to be 1.623 mM and 105 μmol min−1 mg−1, respectively. Human leukemia cell line THP-1 treated with recombinant L-ASNase showed significant morphological changes, and the IC50 of the purified enzyme was found to be 0.8 IU. Moreover, the purified recombinant L-ASNase induced cytotoxic effects on lung adenocarcinoma A549 and colorectal adenocarcinoma Caco-2 cell lines with IC50 of 1.78 IU and 30 IU, respectively.

l -Asparaginases have the potential to inhibit the formation of acrylamide, a harmful toxin formed during high temperature processing of food. A novel bacterium which produces l -asparaginase was screened. Type I l -asparaginase gene from Acinetobacter soli was cloned and expressed in Escherichia coli. The recombinant l -asparaginase had an activity of 42.0 IU mL−1 and showed no activity toward l -glutamine and d -asparagine. The recombinant l -asparaginase exhibited maximum catalytic activity at pH 8.0 and 40°C. The enzyme was stable in the pH ranging from 6.0 to 9.0. The activity of the recombinant enzyme was substantially enhanced by Ba2+, dithiothreitol, and β-mercaptoethanol. The Km and Vmax values of the l -asparaginase for the l -asparagine were 3.22 mmol L−1 and 1.55 IU μg−1, respectively. Moreover, the recombinant l -asparaginase had the ability to mitigate acrylamide formation in potato chips. Compared with the untreated group, the content of acrylamide in samples treated with the enzyme was effectively decreased by 55.9%. These results indicate that the novel type I l -asparaginase has the potential for application in the food processing industry.

Background Hypersensitivity reactions (HSRs) to chemotherapeutic agents have been increasingly documented. Objective The aim of this study was to investigate HSRs due to chemotherapeutics agents in childhood. Methods From January 2007 to June 2019, the patients who were treated for neoplastic diseases in our hospital were evaluated. Patients who developed a HSR to a chemotherapeutic agent were included. Results Fifty-seven patients with 65 reactions (60% anaphylaxis) were evaluated. Escherichia coli asparaginase was responsible for 38 (58.5%) of these 65 reactions. The other agents were polyethylene glycol (PEG)–asparaginase (n = 11), etoposide (n = 7), methotrexate (n = 4), carboplatin (n = 4), and procarbazine (n = 1). Of the 38 patients who had a reaction to E coli–asparaginase, 33 patients received alternative treatment (PEG-asparaginase or Erwinia asparaginase), 3 patients continued with desensitization, and 2 patients underwent bone marrow transplantation. Five patients who had an initial reaction to PEG-asparaginase continued their treatment with Erwinia asparaginase or E coli asparaginase uneventfully. Of 7 patients who had a reaction to etoposide (4 had anaphylaxis), 3 patients continued with desensitization, and 2 patients used the drug with premedication and prolonged infusion. Two patients had anaphylaxis with methotrexate. Treatment was continued with desensitization in 1 patient and methotrexate treatment was discontinued in the other patient. Of the 4 patients with carboplatin hypersensitivity, 2 had anaphylaxis. Desensitization was performed in 2 patients. One patient had procarbazine HSR, drug was given with premedication. Conclusion Among all chemotherapeutic agents reviewed in our study that caused HSRs, asparaginase was the most common culprit agent in children. Most of reactions are immediate type. Many of the patients can take their treatment by drug replacement or desensitization.

L-Asparaginase (L-Asp) is a critical component of chemotherapy for acute lymphoblastic leukemia (ALL). However, toxic effects associated with L-Asp, such as hepatic dysfunction, pancreatitis, hypercholesterolemia, and hyperglycemia, have occurred. In addition, acute pancreatitis is a significant life-threatening adverse event associated with ALL. We describe 2 patients with ALL who had L-Asp-associated pancreatitis (AAP), with one patient presenting with hyperglycemia and the other presenting with hypoglycemia during induction treatment. When octreotide was administered to both of these patients, the clinical findings and laboratory data were improved. AAP was not repeated after treatment with pegylated asparaginase. Although AAP has a high risk of mortality and morbidity in childhood, APP treatment with appropriate agents, such as octreotide, can be successful. (C) 2020 Elsevier Inc.

Acrylamide is a process-induced food toxicant which is formed during heating of food products via Strecker or acrolein pathway. Studies have revealed that acrylamide causes DNA damage, neurotoxicity, genetic toxicity, reproductive toxicity and carcinogenicity. Therefore, efficient approaches should be applied to reduce acrylamide level in foods. However, most of these approaches are not practical, require costly equipment, and cause nutrition loss and decline of sensory properties. In this regard, using specific strains of probiotics especially Lactobacillus has been newly hypothesized and scarcely explored. The mechanism responsible for this activity is associated with the presence of peptidoglycan components particularly carbohydrates and alanine which binds to acrylamide. Another possible mechanism is production of the enzyme asparaginase which converts l -asparagine to l -aspartic acid and ammonia and prevents acrylamide formation. It has been highlighted that some Lactobacillus species (Lactobacillus casei and Lactobacillus reuteri) possess asparaginase genes. Therefore, adding probiotics could provide a good promising approach in reducing the acrylamide in food products. This article reviews the efficacy of probiotics in mitigation of acrylamide in food products.

Extra-nodal natural killer (NK)/T-cell lymphoma is a rare, distinctive clinicopathologic entity with an aggressive clinical course. High expression of multidrug-resistant (MDR) P-gp on NK lymphoma cells renders this disease resistant to anthracycline-based chemotherapy regimens. Over the years, based on retrospective analysis and phase II studies, regimens with L-asparaginase and non-MDR drugs have been used.To evaluate the outcome of patients with extra-nodal NK/T-cell lymphoma treated in a tertiary cancer care center in India from 2012 to 2021.Retrospective, observational, single-center study.Hematolymphoid Department, Tata Memorial Hospital.From January 2010 to December 2021, all consecutive patients with NK/T-cell lymphoma were analyzed.Relapse-free survival and overall survival.A total of 78 patients with a diagnosis of extra-nodal NK/T-cell lymphoma were treated from 2010 to 2021. Amongst them, 56 (71.8%) were male, with a median age of 40 years (15-71 years). The most common presenting symptom was nasal swelling (9%), followed by dysphagia (3.8%). B symptoms were present in 35 patients (45%). Only 5% had HLH anytime during the treatment. One-third of the patients had an ECOG performance status1. Nasal type constituted 80% of the cohort, regional node was involved in 60%, distant node was involved in 28%, 33% had more than 1 extra-nodal site involved, 23% had bulky disease, and 40% had advanced stage (stage 3-4) at presentation. EBV LMP1 was positive in 19 patients (24%). EBERISH was available in 9 patients, of which 7 were positive. Fifteen patients received treatment prior to presentation to our center (CHOP in 10 patients). At our center, the SMILE chemotherapy protocol was administered to 52 patients (66.7%). Consolidative radiation therapy was administered in 43 patients (55.1%). The best radiological response of CR was achieved in 49 patients (62.8%). After a median follow-up of 30 months, 30 (38.5%) patients relapsed and 30 (38.5%) died. Median relapse-free survival was 45 (95%CI: 5.1-84.8) months. Overall survival at 36 months was 53.4% (95%CI: 41.7%-68.4%).The outcome of NK/T-cell lymphoma in our cohort is comparable to the outcomes found in the literature.

Effect of asparaginase was tested in different biscuit and cracker doughs, and wafer batter by changing processing conditions such as enzyme dosage, dough resting time and temperature, mixing speed and time, and mixing procedure of the recipe components. Acrylamide reductions achieved were 96, 80, 54% in rotary cut biscuits, crackers and wire cut cookies, respectively due to high water activity of their dough. Asparaginase did not affect surface color or spread ratio. There was a positive correlation between asparagine content and acrylamide formation except for rotary molded biscuit doughs. No significant decrease was found in rotary molded biscuits because of low water (water activity of 0.70) and high fat content. This indicated that water activity of dough is an important factor for the effectiveness of asparaginase treatment. The results suggested that water activity value exceeding 0.75 is needed in the dough to effectively reduce asparagine, so acrylamide in bakery products.

Acute Lymphoblastic Leukemia (ALL) is the commonest malignancy in childhood with a second incidence peak in adulthood. Improvements in pediatric therapy including the addition of L-asparaginase (L-ASP) have enabled cure rates in excess of 90% to be achieved in children. More recently L-ASP-containing pediatric protocols are being used to treat younger adults with ALL and have improved survival by approximately 2-fold. However, a toxicity associated with L-ASP-containing therapy in ALL is venous thromboembolism (VTE) which is associated with significant morbidity in this patient population and results in interruptions in L-ASP therapy that can impact on survival outcomes. The incidence of VTE among adult patients with ALL receiving L-ASP containing therapy has been reported to be as high as 43%. Despite this, there is a lack of evidence-based recommendations for VTE prophylaxis in this clinical context; low-molecular weight heparin (LMWH) and/or AT replacement have mostly been used. The low-quality data and inconveniences associated with these VTE prophylaxis regimens highlight the need to evaluate alternatives such as direct oral anticoagulants for the prevention of L-ASP-associated VTE in ALL. This narrative will review the body of evidence on primary thromboprophylaxis in adult patients with ALL receiving L-ASP containing therapy.

The genome sequence of the hyperthermophilic archaeon Thermococcus kodakarensis contains two putative genes, TK1656 and TK2246, annotated as l -asparaginases. TK1656 has been reported previously. The current report is focused on TK2246, a plant-type l -asparaginase, which consists of 918 nucleotides corresponding to a polypeptide of 306 amino acids. The gene was cloned, expressed in Escherichia coli and the purified gene product was used to determine the properties of the recombinant enzyme. TK2246 was optimally active at 85 °C and pH 7.0 with a specific activity of 767 μmol min−1 mg−1 towards l -asparagine. The enzyme exhibited a 10% activity towards d -asparagine as compared to 100% against l -asparagine. No detectable activity was observed towards l - or d -glutamine. Half-life of the enzyme was nearly 18 h at 85 °C. TK2246 exhibited apparent Km and Vmax values of 3.1 mM and 833 μmol min−1 mg−1, respectively. Activation energy of the reaction, determined from the Arrhenius plot, was 28.3 kJ mol−1. To the best of our knowledge, this is the first characterization of a plant-type l -asparaginase from class Thermococci of phylum Euryarchaeota.

Chaperones are a diverse class of molecules known for increasing thermo-stability of proteins, preventing protein aggregation, favoring disaggregation, increasing solubility and in some cases imparting resistance to proteolysis. These functions can be employed for various biotechnological applications including point of care testing, nano-biotechnology, bio-process engineering, purification technologies and formulation development. Here we report that the N-terminal domain of Pyrococcus furiosusl-asparaginase, (NPfA, a protein chaperone lacking α-crystallin domain) can serve as an efficient, industrially relevant, protein additive. We tested the effect of NPfA on substrate proteins, ascorbate peroxidase (APX), IgG peroxidase antibodies (I-HAbs) and KOD DNA polymerase. Each protein not only displayed increased thermal stability but also increased activity in the presence of NPfA. This increase was either comparable or higher than those obtained by common osmolytes; glycine betaine, sorbitol and trehalose. Most dramatic activity enhancement was seen in the case of KOD polymerase (∼ 40 % increase). NPfA exerts its effect through transient binding to the substrate proteins as discerned through isothermal titration calorimetry, dynamic light scattering and size exclusion chromatography. Mechanistic insights obtained through simulations suggested a remodeled architecture and emergence of H-binding network between NPfA and substrate protein with an effective enhancement in the solvent accessibility at the active site pocket of the latter. Thus, the capability of NPfA to engage in specific manner with other proteins is demonstrated to reduce the concentration of substrate proteins/enzymes required per unit operation. The functional expansion obtained through our finding establishes NPfA as a novel class of ATP-independent molecular chaperone with immense future biotechnological applications.

Venous thromboembolism is a common complication of asparaginase-based chemotherapy regimens for the treatment of acute lymphoblastic leukemia. Thrombosis associated with asparaginase administration poses a number of specific and often clinically challenging management decisions. This review provides guidance on the prevention and treatment of thrombosis associated with asparaginase in adults including discussions on antithrombin repletion, pharmacologic thromboprophylaxis, cerebral venous thrombosis, and therapeutic anticoagulation.

Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Therapies for pediatric ALL have improved such that more than 80% of patients survive to 5 years post-therapy, and most survive to adulthood. These ALL patients experience long-term side effects that permanently affect their quality of life, with bone loss and reduced longitudinal growth being the most common skeletal complications. To determine the effects of the chemotherapeutic agents used in ALL induction therapy on bone density and longitudinal growth in mice, we treated juvenile mice with doxorubicin, dexamethasone, vincristine, l-asparaginase, or combination therapy. At adulthood, mice were culled and bones collected and scanned by micro-computed tomography (micro-CT). Mice that received doxorubicin and combination therapy exhibited reduced longitudinal growth and significant reductions in trabecular bone volume, trabecular thickness, and trabecular number, with increased trabecular separation. Mean cortical thickness, cortical area, marrow area, endocortical perimeter, and polar moment of inertia were significantly reduced by doxorubicin and combination therapy. Vincristine treatment significantly decreased trabecular bone volume, trabecular number, and increased trabecular separation but had no effects on cortical bone. Dexamethasone treatment increased trabecular bone separation, cortical marrow area, and cortical bone periosteal perimeter. Mice treated with l-asparaginase did not have any bone phenotypes. In conclusion, these data indicate that the majority of the chemotherapy agents used in induction therapy for pediatric ALL have long-term effects on bone in mice. A single dose of doxorubicin in juvenile mice was sufficient to cause the majority of the bone phenotypes, with combination therapy intensifying these effects.

Asparaginase (ASNase) causes pancreatitis in approximately 10% of leukemia patients, and the mechanisms underlying this painful complication are not known. ASNase primarily depletes circulating asparagine, and the endogenously expressed enzyme, asparagine synthetase (ASNS), replenishes asparagine. ASNS was suggested previously to be highly expressed in the pancreas. In this study, we determined the expression pattern of ASNS in the pancreas and the mechanism for increased pancreatic ASNS abundance. Compared with other organs, ASNS was highly expressed in both the human and mouse pancreas, and, within the pancreas, ASNS was present primarily in the acinar cells. The high baseline pancreatic ASNS was associated with higher baseline activation of protein kinase R-like endoplasmic reticulum kinase (PERK) signaling in the pancreas, and inhibition of PERK in acinar cells lessened ASNS expression. ASNase exposure, but not the common pancreatitis triggers, uniquely up-regulated ASNS expression, indicating that the increase is mediated by nutrient stress. The up-regulation of acinar ASNS with ASNase exposure was owing to increased transcriptional rather than delayed degradation. Knockdown of ASNS in the 266-6 acinar cells provoked acinar cell injury and worsened ASNase-induced injury, whereas ASNS overexpression protected against ASNase-induced injury. In summary, ASNS is highly expressed in the pancreatic acinar cells through heightened basal activation of PERK, and ASNS appears to be crucial to maintaining acinar cell integrity. The implications are that ASNS is especially hardwired in the pancreas to protect against both baseline perturbations and nutrient deprivation stressors, such as during ASNase exposure. Keywords: Leukemia, Asparaginase-Associated Pancreatitis, Asparagine Synthetase, PERK Signaling

Directed evolution methodologies have been used as promising strategies for improving the catalytic properties of many existing enzymes. In the presented work, this approach was applied to improve the enzyme activity of l -asparaginase I obtained from Bacillus megaterium H-1. After two rounds of error-prone polymerase chain reaction (epPCR) and two generations of sequential DNA shuffling, all of 5 different mutants showed a significant increase in the enzyme activity of l -asparaginase I, ranging from 6.27 to 22.78 IU/mL. Among these mutants, D-9B and DD-12G displayed the relatively high catalytic activity, which were 20.22-fold and 21.33-fold higher than the wild-type enzyme (WT), respectively. Furthermore, the catalytic efficiency (kcat/Km) of D-9B and DD-12G were also improved, which were 132.73 min−1mM−1 and 146.39 min−1mM−1, respectively, in comparison to that of WT (3.39 min−1mM−1). In addition, mutant DD-12G showed tolerance toward wider range of pH values and higher temperatures than its WT counterpart. Homology modeling of above two mutants reflected a reduction of hydrogen bonds and an introduction of flexible residues in the loops near the active catalytic site Thr15. These changes contributed to the flexibility of loops, which may lead to further enhancement in catalytic efficiency. Results also showed that approximately 88.5% (0.978 mg/kg) acrylamide could be removed from mutant DD-12G pre-treated fried potato chips. This study clearly shows that directional evolution methods can indeed be utilized to improve the activity of l -asparaginase, which could also provide research basis for future application in food industry.

Cell-free protein synthesis has emerged as a promising platform for the production of therapeutic proteins due to its inherently open reaction environment, flexible reaction conditions and rapid protein synthesis capabilities. In recent years, lyophilized cell-free systems have widened the application space of cell-free technology by improving reagent stability outside of cold-chain storage. Current embodiments of the system, however, demonstrate poor stability at elevated temperatures. Lyoprotectants have long been recognized for the ability to preserve the activity of biological molecules during drying processes, but the application of this technology to lyophilized cell-free systems has been limited and has failed to address the negative effects that such lyoprotectants may have on cell-free systems. Here, several lyoprotected, lyophilized cell-free protein synthesis systems are demonstrated using antiplasticized sugar glasses as lyoprotectants, showing significant improvement over standard lyophilized systems or trehalose-preserved systems. Furthermore, we demonstrate for the first time, preservation and therapeutic expression, specifically of FDA-approved crisantaspase, from a truly single-pot lyophilized, endotoxin-free, cell-free protein synthesis system, exemplifying the potential for on-site therapeutic synthesis.

Lymphoma is the 3rdmost common neoplasm in domestic ferrets (Mustela putorius furoi). The objective of this study was to describe case demographics and outcomes of ferrets diagnosed and treated for lymphoma. Forty-four ferrets from two institutions were retrospectively evaluated for case demographics, subjective response to treatment, date of last known status (lost to follow-up [LTFU], euthanized, died) and survival time (ST; days between diagnosis and last known status). Multiagent chemotherapy protocols included modified cyclophosphamide, vincristine, prednisolone (COP) ± l -asparaginase ( l -ASP); modified COP, doxorubicin, l -ASP; Tufts no-IV ± l -ASP; prednisolone, cyclophosphamide ± l -ASP; prednisolone and l -ASP; prednisolone, vincristine and l -ASP; prednisolone, vinblastine and l -ASP; cyclophosphamide and l -ASP; cyclophosphamide, vincristine ± l -ASP. Prednisolone, chlorambucil or cyclophosphamide was used as single-agent therapies in eight cases. The median ST (MST) for ferrets with a known date of death (DOD) was 126 days (n = 37, range 3–1199 days) overall ST. Ferrets treated with modified COP ± additional chemotherapeutics had a MST of 429 days (n = 15, range 35–1199 days); all had a known DOD. The MST for all seven ferrets that received the Tufts no-IV protocol was 86 days (range 19–888 days) with 1/7 having a known DOD at 73 days and the remainder being LTFU. This manuscript provides descriptive data that may be beneficial for veterinarians having treatment discussions with their clients. Due to the inability to control for lymphocyte cell size, staging and treatment heterogeneity, a comparison between protocols was not performed.

Background Treatment of acute lymphoblastic leukemia (ALL) in adolescent and young adult (AYA) patients using traditional adult chemotherapy protocols give low overall survival (OS) rates. Data are growing regarding the use of pediatric-inspired chemotherapy protocols in AYA patients with improvement in OS. Patients and Methods To assess efficacy and tolerability of using a pediatric-inspired protocol in AYA patients, we initiated our local prospective trial using a modified version of the Children’s Cancer Group 1900 protocol for newly diagnosed high-risk Philadelphia chromosome-negative ALL patients. Results A total of 40 patients were enrolled in the study (from 2015 to 2018). The median age was 18 years (range, 14-34 years). The complete remission rate after induction was 37 patients [93%] and after a median follow-up of 5 years, OS, disease-free survival (DFS), and event-free survival were 75%, 72%, and 60%, respectively. Use of this protocol was well tolerated with manageable toxicities. Pegylated asparaginase was given to all patients during the induction phase and was well tolerated. Conclusion The use of a pediatric-inspired protocol for high-risk AYA ALL patients was effective and well tolerated with improvement in OS and DFS compared with historical data using adult protocols in such populations.

The present paper describes efficient immobilization of L-glutaminase free L-asparaginase for developing a new therapeutic system for anticancer therapy. L-asparaginase (L-ASNase) was covalently immobilized on the functionalized aluminum oxide nanoparticles (AONP) and titanium oxide nanoparticles (TONP). The nano-bioconjugates (AONP-ASNase and TONP-ASNase) were characterized by scanning electron microscope (SEM), Fourier-transform infrared spectroscopy (FTIR) and UV–Vis spectral analysis that revealed the successful immobilization. The nano-bioconjugates were optimally active at pH 7.0 and 40 °C. TONP-ASNase activity was enhanced in the presence of NH4+ (160%) and Mn2+ (165%) while AONP-ASNase bioconjugates showed increased relative activity with ethyl acetate (142%) and toluene (160%). The nano-bioconjugates displayed excellent reusability and maintained >90% average activity after nine successive cycles. Maximum cytotoxicity (61%) was noticed with AONP-ASNase (10 μg/ml) against human leukemia MOLT-4 cells. Regarding kinetic values, AONP-ASNase showed better affinity (Km 1.9 μmol) to L-asparagine as compared to free L-ASNase. After 23 days storage at 37 °C, bioconjugates retained 40% residual activity while free L-ASNase was completely deactivated. Thermodynamic characterization revealed higher conversion rate of the E-S complex in case of nano-bioconjugates.

This study aimed to produce and characterize thermostable l -asparaginase from Bacillus amyloliquefaciens MKSE, a newly screened strain from Korean fermented soybean paste. The isolated MKSE was identified by its morphological, biochemical, and physiological properties and 16S rRNA sequence. Its l -asparaginase II-like gene was expressed in Escherichia coli BL21 (DE3), by using the pET-28a (+) vector. The produced l -asparaginase (specific activity of 4.75 IU/mg) was purified by a one-step procedure, using nickel-affinity chromatography, and its molecular weight of 38.2 kDa was determined by gel electrophoresis. Its optimum pH and temperature were 8.5 and 65 °C, respectively. The enzyme was highly stable in the pH range of 3.0–11.0 for 6 h incubation, and the enzyme retained about 57% of its activity at 95 °C for 6 h. Its activity was activated in the presence of Mn2+, Mg2+, and Fe2+ while it was inhibited by Zn2+, Cu2+, PMSF, and SDS. Its Km was 1.5 mM, and its Vmax was 136.3 IU/mg. The results of this study reveal that MKSE l -asparaginase has a great potential to be used in various industries, due to its stability at high temperature and over a wide pH range.

Acrylamide has received worldwide attention due to its existence in commonly consumed foods. L-asparaginase reduces acrylamide formation in foods by hydrolyzing available L-asparagine. Herein, L-asparaginase (Ps-ASNase II) of Pseudomonas sp. PCH182 was expressed in Escherichia coli (E. coli), purified, and evaluated for acrylamide reduction in food samples. The monomeric 37 kDa Ps-ASNase II protein was purified to homogeneity with a 70 % yield. The enzyme was active at a wide pH range (5.0-11.0) and temperature (10-80 °C) with optimum activity at 45 °C in 50 mM Tris-HCl (pH 8.5) after 10 min. The K

The results of treatment of adolescents and adults with acute lymphoblastic leukemia (ALL) remain unsatisfactory. Pediatric-inspired treatments seem to be related with better outcomes. 126 adolescent and adult patients with ALL were treated in a 37-year period with a pediatric inspired combined chemotherapy (PICC) schedule, delivered on an outpatient basis and based on the St. Jude´s TOTAL XI pediatric protocol employing vincristine, prednisone, asparaginase, daunorubicin, etoposide, cytarabine, methotrexate, mercaptopurine and triple intrathecal therapy. 80 % of patients were able to receive the initial seven-week period of induction / consolidation fully as outpatients and 77 % achieved a complete remission. In adolescents and young adults (AYAs) the median probability of overall survival (OS) was 44 months, whereas the 5-year OS was 48 %. In adults, the median probability of OS was 24 months, and the 5-year OS was 32 %. Patients with T-cell ALL did significantly worse than those with a B cell phenotype (OS at 5 years 17 versus 40 %, respectively). These figures are better than those informed in our country employing more aggressive, in-hospital schedules such as the hyper-CVAD. We found that, in AYAs and adult patients with ALL, the use of an asparaginase-containing PICC delivered on an outpatient basis renders acceptable results, better than those obtained in similar socioeconomic circumstances employing adult-oriented schedules. Additional studies are needed to assess the usefulness of these PICC treatments in adult individuals with ALL treated in underprivileged circumstances, such as those prevailing in LMIC.

Purpose/Objective(s) This study evaluated the survival benefit of asparaginase (ASP)-based versus non-ASP-based regimens in a real-world cohort of patients with early-stage extranodal nasal-type NK/T-cell lymphoma (ENKTCL). Materials/Methods In the modern era, we identified 376 patients who received combined radiotherapy with either ASP-based (asparaginase, platinum and gemcitabine, n = 286) or non-ASP-based (platinum and gemcitabine, n = 90) regimens. The patients were stratified into low-, intermediate- and high-risk groups using the early stage-adjusted nomogram-revised risk index (ES-NRI). Overall survival (OS) and distant metastasis (DM)-free survival (DMFS) between the chemotherapy regimens were compared using inverse probability of treatment weighting (IPTW) and multivariable analyses. Results ASP-based (versus non-ASP-based) regimens significantly improved 5-year OS (84.5% vs. 73.2%, P = 0.021) and DMFS (84.4% vs. 74.5%, P = 0.014) for intermediate- and high-risk patients, but not for low-risk patients. Moreover, ASP-based regimens decreased DM, with 5-year cumulative DM rate of 14.9% for ASP-based regimens compared with 25.1% (P = 0.014) for non-ASP-based regimens. The survival benefit of ASP-based regimens remained consistent after adjusting the confounding variables by IPTW and multivariate analyses. Additional sensitivity analyses confirmed these results. Conclusion These findings provide an evidence supporting ASP-based regimens as a first-line combined-modality treatment for intermediate- and high-risk early-stage ENKTCL.

Summary Contemporary paediatric clinical trials have improved 5-year event-free survival above 85% and 5-year overall survival above 90% in B-cell acute lymphoblastic leukaemia (ALL) in many study groups, whilst outcomes for T-cell ALL are still lagging behind by 5–10% in most studies. Several factors have contributed to this discrepant outcome. First, patients with T-cell ALL are generally older than those with B-cell ALL and, therefore, have poorer tolerance to chemotherapy, especially dexamethasone and asparaginase, and have increased risk of extramedullary relapse. Second, a higher proportion of patients with B-cell ALL have favourable genetic subtypes (eg, ETV6–RUNX1 and high hyperdiploidy), which confer a superior outcome compared with favourable subtypes of T-cell ALL. Third, T-cell ALL blasts are generally more resistant to conventional chemotherapeutic drugs than are B-cell ALL blasts. Finally, patients with B-cell ALL are more amendable to available targeted therapies, such as Philadelphia chromosome-positive and some Philadelphia chromosome-like ALL cases to ABL-class tyrosine kinase inhibitors, and CD19-positive and CD22-postive B-cell ALL cases to a variety of immunotherapies. Several novel treatments under investigation might narrow the gap in survival between T-cell ALL and B-cell ALL, although novel treatment options for T-cell ALL are limited.

Asparaginase catalyzes the conversion of asparagine into aspartic acid and ammonia. The enzyme has various industrial applications and it is considered as an anticancer drug for treatment of certain leukemias. In the current study, production of asparaginase was investigated by Yarrowia lipolytica as well as optimized its production and determined its molecular characteristics by in silico analysis. Y. lipolytica DSM3286 produced 17.14 U/ml of asparaginase in flask culture. Optimization of asparaginase production was done by response surface methodology and the enzyme production increases up to 102.85 U/ml. The enzyme production reached 210 U/ml in a bioreactor which is 12-fold more than flask culture containing non-optimized medium. Asparaginase gene of Y. lipolytica was identified and isolated on the basis of comparison with asparaginase gene sequences of other microorganisms. The gene has 981 nucleotides and its protein has 326 amino acids. According to in silico analysis, the secondary structure of the enzyme is composed of 9 α-helixes and 11 β-sheets. Y. lipolytica produces type II asparaginase with high affinity for asparagine which is a suitable eukaryotic asparaginase for treatment of hematopoietic cancers. Hence, Y. lipolytica could be recommended as a new eukaryotic microbial source for the production of this important therapeutic enzyme.

During the treatment of acute lymphoblastic leukemia (ALL) with asparaginase (ASN) there is an accumulation of ammonia in the body as result of asparagine hydrolysis. This accumulation known as hyperammonemia is one of the main side-effects of this therapy. To avoid hyperammonemia is urgent to develop new strategies for ammonia retention. Herein is presented the immobilization of ASN into bovine serum albumin/poloxamer 407 (BSA/Pol407) nanoparticles. The ability of the developed nanoparticles to hydrolyze asparagine while retaining the forming ammonia is also explored. Different percentages of ASN were entrapped into BSA nanoparticles coated with Poloxamer 407 and were prepared by high-pressure homogenization. The nanoparticles were characterized regarding their physico-chemical properties, stability, capacity to retain ammonia and safety using zebrafish embryos as an in vivo model of toxicity. The BSA/ASN25%/Pol407 nanoparticles were selected as the best formulation to hydrolyze asparagine using the lowest nanoparticle concentration. These nanoparticles presented physical characteristics suitable for an intravenous application and were capable to retain the forming ammonia decreasing the negative effect of free ASN on zebrafish survival. These nanoparticles could potentially be used to prevent hyperammonemia during ALL treatment with ASN., This study was supported by FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01- 0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145- FEDER-000004) and Nanotechnology Based Functional Solutions (NORTE-01-0145-FEDER-000019) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. We also acknowledge the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through Fundação para a Ciência e a Tecnologia (FCT) and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI). Marisa P. Sárria was supported by Marie Curie COFUND funding from the European Union’s 7th Framework Programme for research, technological development and demonstration under grant agreement 600,375. Artur Ribeiro and Ana Tinoco thanks FCT for funding the scholarships with the references SFRH/BPD/98388/2013, SFRH/BD/ 114035/2015, respectively., info:eu-repo/semantics/publishedVersion

Asparaginase is an integral component of acute lymphoblastic leukemia (ALL)

Co-encapsulation of multiple therapeutic drugs in a single nanocarrier has the potential to enable synergistic interactions, increase drug efficacy, and reduce side effects. The enzyme l-asparaginase and the small molecule drug etoposide have a known synergistic effect against selected cancer types. However, both drugs differ significantly in size, molecular weight, and solubility, which often results in challenges when a simultaneous delivery is required. In this study, we present the co-encapsulation of a large hydrophilic enzyme l-asparaginase and the small hydrophobic drug etoposide into a biodegradable, biocompatible, and acid-responsive dextran-based nanoparticle system. These dual drug-loaded nanoparticles show an excellent cellular uptake in chronic myeloid leukemia (CML) K562 cells and a stepwise release of the cytotoxic payloads in a pH-dependent manner. In activity tests, the dual drug-loaded formulation has shown a significant effect on cell viability (down to 31%) compared to those incubated only with l-asparaginase (92%) or etoposide (82%) at a particle concentration of 125 μg∙mL

The asparaginase II pathway consists of an asparagine transaminase [ l -asparagine + α-keto acid ⇆ α-ketosuccinamate + l -amino acid] coupled to ω-amidase [α-ketosuccinamate + H2O → oxaloacetate + NH4+]. The net reaction is: l -asparagine + α-keto acid + H2O → oxaloacetate + l -amino acid + NH4+. Thus, in the presence of a suitable α-keto acid substrate, the asparaginase II pathway generates anaplerotic oxaloacetate at the expense of readily dispensable asparagine. Several studies have shown that the asparaginase II pathway is important in photorespiration in plants. However, since its discovery in rat tissues in the 1950s, this pathway has been almost completely ignored as a conduit for asparagine metabolism in mammals. Several mammalian transaminases can catalyze transamination of asparagine, one of which – alanine-glyoxylate aminotransferase type 1 (AGT1) – is important in glyoxylate metabolism. Glyoxylate is a precursor of oxalate which, in the form of its calcium salt, is a major contributor to the formation of kidney stones. Thus, transamination of glyoxylate with asparagine may be physiologically important for the removal of potentially toxic glyoxylate. Asparaginase has been the mainstay treatment for certain childhood leukemias. We suggest that an inhibitor of ω-amidase may potentiate the therapeutic benefits of asparaginase treatment.

Acute lymphoblastic leukemia (ALL) is a malignancy of lymphoid progenitor cells occurring at an annual incidence rate of approximately 1.1–2.1 per 100,000 person-years globally. Approximately 40% of annual ALL cases occur in adults, yet estimated 5-year overall survival (OS) rates are about 30%–40% in adults (and vary broadly by age) compared with 90% in children. Although the addition and/or intensification of asparaginase as a key treatment strategy for pediatric ALL is well recognized, further research is needed to clarify the benefit/risk ratio in adult patients with ALL. This review emphasizes the importance of efficient management of adverse events to increase asparaginase efficacy, and explores novel strategies for optimizing asparaginase treatment, including new formulations of asparaginase, pharmacokinetic-based dosing, and pharmacogenetic profiling. Upcoming results of adult ALL trials should further clarify the role of asparaginase, building on the results of the large NOPHO 2008, CALGB 10403, GRAALL-2005, GMALL 07/2003, and UKALL14 trials.

There is an increasing interest in using l -asparaginase in medical fields and food processing industries. Enzyme immobilization is an attractive field to improve l -asparaginase activity and stability. Graphene oxide (GO) is a promising candidate for enzyme immobilization due to its large specific surface area. In this study, GO was first functionalized with l -aspartic acid (GO-Asp), and then l -asparaginase was immobilized on the GO-Asp either physically or through chemical conjugation. A significant enzyme loading was achieved through covalent immobilization (100% immobilization efficiency). Stability of free and the immobilized l -asparaginase was examined at various temperatures (20–60 °C) and pH (5–9). The covalently immobilized l -asparaginase showed higher enzyme activity than free enzyme at pH 8 with the maximum recovered activity of 100%, 90.5% and 40.6% after 24 h of incubation at 20 °C, 40 °C and 60 °C, respectively. In addition, the covalently immobilized l -asparaginase on GO-Asp showed 42% recovered activity after eight continuous reaction cycles at 60 °C. The kinetic parameters of the immobilized and free enzyme were also calculated, indicating no significant changes in the enzyme affinity through covalent conjugation. The results clearly reflect the suitability of GO-Asp as a nanosheet support for l -asparaginase loading as well as its usage in future industrial applications.

Acrylamide is a potent carcinogen and neurotoxin formed by the Maillard reaction when l-asparagine reacts with starch at high temperature. It is formed in food materials mainly deep fried and bakery products. Enzymatic pretreatment of these food products with asparaginase enzyme leads to reduction in acrylamide. However, enzymatic process is quite expensive due to high cost, low catalytic efficiency as well as problem with enzyme reusability. Present work deals with these problems by exploring l-asparaginase from Bacillus aryabhattai. Asparaginase enzyme was immobilized on APTES modified magnetic nanoparticles. It was found to be more than three-fold increase their thermal stability from free enzyme and retained 90% activity after fifth cycle. The immobilized enzyme also showed better affinity towards its substrate. During pretreatment of asparagine in a starch-asparagine food model system and it was clearly demonstrated that asparaginase nanoconjugates had reduced the formation of acrylamide by more than 90% within 30 min.

Cerium selenium nanobiocomposites are novel lung cancer drug as they possess combined anti-cancer property of nanocomposite with l -asparaginase working in synergetic manner. Cerium selenium nanobiocomposites were synthesized using simple co-precipitation method. The size of the nanocomposite was found to be in the range 60–90 nm. Maximum absorption was observed using UV spectrum in the range of 350–490 nm. The nanobiocomposites was characterized using H-NMR and FTIR analysis it was found that secondary alkyl, allylic carbon, monosubstituted alkenes and sp2 hybridized C H bonds of alkenes were involved in binding of cerium and selenium nanoparticles with l -asparaginase for the formation of cerium selenium nanobiocomposite. The spherical shape of the cerium selenium nanobiocomposites were confirmed using SEM. Anticancer activity was checked by performing MTT assay resulting in 70.84% and 48.78% toxicity for maximum concentration of 1000 (μg/ml) and IC50 concentration of 125 (μg/ml) respectively on A549 lung cancer cell line using fluorescent microscopic analysis.

Type II l‑asparaginase (l‑ASNase) is an FDA approved enzyme drug with extensive applications for treatment of certain blood cancers. However, the therapeutic efficiency of this enzyme is hampered by its undesirable glutaminase activity. Given the pivotal role of this enzyme against cancer, designing engineered mutants with diminished glutaminase activity would be of great therapeutic interest. To this end, N248S mutation was selected as the potential mutation with beneficial effects. Various in silico analyses including MD simulation, molecular docking and QMMM studies were performed to assess the effects of N248S mutation on the activity of the enzyme. Thereafter, this mutation along with N248A, N248V and N248T mutations as controls were exerted in l‑ASNase gene. The results from in silico analyses and experimental efforts indicated that N248S mutation is associated with the suitable l‑ASNase activity, while the glutaminase activity is disturbed due to impaired interactions. It has been shown that glutamine turnover was affected much more strongly than asparagine hydrolysis. The approach of exploiting in silico tools to design mutated enzymes lead to staggering time and cost reduction. Following this strategy, we have designed a mutant l‑ASNase with diminished glutaminase activity, which could be of interest for improved biomedical applications.

Bioactive compounds from endophytes have been used to treat various diseases. In the present study, L-Asparaginase producing endophytes were isolated from Ocimum tenuiflorum (Tulasi) from NIT Warangal, Telangana, India to treat Acute Lymphoblastic Leukemia (ALL) in which L-Asparagine (L-Asn) deamination plays a vital role in ALL treatment. 20 (bacteria and fungi) out of 35 endophytes have been screened for L-Asparaginase production using rapid plate assay technique, in which four strains produced high amounts of L-Asparaginase. 16 s Ribosomal RNA sequencing studies were performed, Bacillus stratosphericus organism was identified, and purified L-Asparaginase sequence has been tailored using MALDI/TOF (Applied Biosystems). The homology model was developed by using MODELLER 9.15v as the endophyte lacks crystal structure of L-Asparaginase enzyme and validated by dint of quality index tools. Docking studies were performed using iGemdock 2.1v. In comparison, free energy binding efficiency of receptor towards L-Asparagine (L-Asn) is good with lesser energy -71.6 kcal/mol in comparison to L-Glutamine (L-Gln) having -67.7 kcal/mol. In order to find the stability of the docked complexes in dynamics environment, molecular dynamics and simulation studies were performed using GROMACS V4.6.5. The trajectory analysis for 10 ns shows the better RMSD, RMSF, Rg and average number of hydrogen bonds for complex 1 (L-Asparaginase + L-Asn docked complex). Hence, complex 1 was found to be more stable than Complex 2 (L-Asparaginase + L-Gln docked complex).

l-asparagine essentially regulates growth and proliferation of cancer cells. l-asparaginase is an anti-cancer enzyme that deprives the cancer cells of l-asparagine. The purpose of this study was to explore the mechanism of a novel l-asparaginase from Pseudomonas fluorescens on l-asparagine deprivation mediated anti-proliferation, apoptosis in human gastric adenocarcinoma cells and to evaluate inhibition of angiogenesis. We observed that, the presence of extracellular l-asparagine was essential for the growth of AGS cells. l-asparagine deprivation by l-asparaginase induced metabolic stress, cytotoxicity and apoptosis by G0 phase cell-cycle arrest, modulated the mitochondrial membrane integrity, accelerated caspase-3 activation and instigated DNA damage. The RT-PCR analysis of pro-apoptosis genes: bak1, bax, bbc3, bik, pmaip1, bnip3l, apaf1, casp3, casp7 and casp9 were significantly higher (P 0.05), while anti-apoptotic markers xiap, bid, mcl1, and death receptor genes tnf and tradd were significantly down-regulated (P 0.05). Additionally, higher protein expressions of p53, caspase-3 and TEM analysis showing modulations in mitochondria confirmed intrinsic apoptosis pathway. The enzyme impeded tumor progression through inhibition of cell migration and vascular remodelling of endothelial cells. Our findings suggests that the action of l-asparaginase alters mitochondrial membrane permeability and auxiliary activates intrinsic apoptosis. Therefore, this mechanistic approach might be considered as a targeted enzymotherapy against gastric adenocarcinoma.

l -asparaginase (EC 3.5.1.1) needs to endure high temperatures during applications in food processing. We here improved the thermal stability of Bacillus subtilis 168 l -asparaginase by regulating both the kinetic and thermodynamic states of the enzyme. According to the molecular dynamics simulation for l -asparaginase, 10 flexible residues potentially affecting the kinetic stability were individually subjected to saturation mutagenesis, generating two highly stabilized mutants, S180N and D289T. As predicted by Discovery Studio, eight residues of l -asparaginase formed multiple unfavourable charge–charge interactions adversely affecting thermodynamic stability. Single-site saturation mutagenesis on these residues resulted in two thermodynamically stabilized mutants, E260F and E292S. Using these mutants, we constructed a further stabilized quadruple mutant NTFS (S180N/D289T/E260F/E292S). Compared with the wild-type enzyme, NTFS exhibited an 8.1-fold increase in half-life at 65 °C and a 5.56 °C increase in melting temperature. NTFS also showed a substantial increase in the transition state energy barrier (ΔΔGtransition = 5.27 KJ/mol) and a clear decrease in folding free energy (ΔΔGfolding = −19.12 kcal/mol) relative to the wild-type enzyme. These results indicated that simultaneous regulation of both the kinetic and thermodynamic states of an enzyme represents an efficient strategy for enhancing thermal stability. The highly stable NTFS described here might be beneficial for industrial application.