Exploring the potential of asparagine restriction in solid cancer treatment: recent discoveries, therapeutic implications, and challenges.

Asparagine is a non-essential amino acid crucial for protein biosynthesis and function, and therefore cell maintenance and growth. Furthermore, this amino acid has an important role in regulating several metabolic pathways, such as tricarboxylic acid cycle and the urea cycle. When compared to normal cells, tumor cells typically present a higher demand for asparagine, making it a compelling target for therapy. In this review article, we investigate different facets of asparagine bioavailability intricate role in malignant tumors raised from solid organs. We take a comprehensive look at asparagine synthetase expression and regulation in cancer, including the impact on tumor growth and metastasis. Moreover, we explore asparagine depletion through L-asparaginase as a potential therapeutic method for aggressive solid tumors, approaching different formulations of the enzyme and combinatory therapies. In summary, here we delve into studies about endogenous and exogenous asparagine availability in solid cancers, analyzing therapeutic implications and future challenges.

Chemical composition and sensory profiling of coffees treated with asparaginase to decrease acrylamide formation during roasting.

Acrylamide is an amide formed in the Maillard reaction, with asparagine as the primary amino acid precursor. The intake of large amounts of acrylamide has induced genotoxic and carcinogenic effects in hormone-sensitive tissues of animals. The enzime asparaginase is one of the most effective methods for lowering the formation of acrylamide in foods such as potatoes. However, the reported sensory outcomes for coffee have been unsatisfactory so far. This study aimed to produce coffees with reduced levels of acrylamide by treating them with asparaginase while retaining their original sensory and bioactive profiles. Three raw samples of Coffea arabica, including two specialty coffees, and one of Coffea canephora were treated with 1000, 2000, and 3000 ASNU of the enzyme. Asparagine and bioactive compounds (chlorogenic acids-CGA, caffeine, and trigonelline) were quantified in raw and roasted beans by HPLC and LC-MS, while the determination of acrylamide and volatile organic compounds was performed in roasted beans by CG-MS. Soluble solids, titratable acidity, and pH were also determined. Professional cupping by Q-graders and consumer sensory tests were also conducted. Results were analyzed by ANOVA-Fisher, MFA, PCA and Cluster analyses, with significance levels set at p ≤ 0.05. Steam treatment alone decreased acrylamide content by 18.4%, on average, and 6.1% in medium roasted arabica and canefora coffees. Average reductions of 32.5-56.0% in acrylamide formation were observed in medium roasted arabica beans when 1000-3000 ASNU were applied. In the canefora sample, 59.4-60.7% reductions were observed. However, steam treatment primarily caused 17.1-26.7% reduction of total CGA and lactones in medium roasted arabica samples and 13.9-22.0% in canefora sample, while changes in trigonelline, caffeine, and other evaluated chemical parameters, including the volatile profiles were minimal. Increasing enzyme loads slightly elevated acidity. The only sensory changes observed by Q-graders and or consumers in treated samples were a modest increase in acidity when 3000 ASNU was used in the sample with lower acidity, loss of mild off-notes in control samples, and increased perception of sensory descriptors. The former was selected given the similarity in chemical outcomes among beans treated with 2000 and 3000 ASNU loads.

Free amino acid profile in Mimosa scabrella honeydew honey from Brazil and chemometric analysis for geographical discrimination.

In this study, twenty free amino acids (FAA) were investigated in samples of bracatinga (Mimosa scabrella) honeydew honey (BHH) from Santa Catarina (n = 15) and Paraná (n = 13) states (Brazil), followed by chemometric analysis for geographic discrimination. The FAA determination was performed by gas chromatography-mass spectrometry (GC-MS) after using a commercial EZ:faast™ kits for GC. Eight FAA were determined, being proline, asparagine, aspartic and glutamic acids found in all BHH, with significant differences (p < 0.05). In addition, with the exception of proline, the others FAA (asparagine, aspartic and glutamic) normally showed higher concentrations in samples from Santa Catarina state, being that in these samples it was also observed higher FAA sums (963.41 to 2034.73 mg kg-1) when compared to samples from Paraná state. The variability in the results did not show a clear profile of similarity when the heatmap and hierarchical grouping were correlated with the geographic origin and the concentration of eight determined FAA. However, principal component analysis (PCA) demonstrated that serine, asparagine, glutamic acid, and tryptophan were responsible for the geographic discrimination among samples from Santa Catarina and Paraná states, since they were the dominant variables (r > 0.72) in the PCA. Therefore, these results could be useful for the characterization and authentication of BHH based on their FAA composition and geographic origin.

Aplicação de ferramentas de quimioinformática na comparação da estrutura tridimensional da dihidrofolato redutase expressa em diferentes organismos / Application of cheminformatics tools for comparing dihydrofolate reductase's tridimensional structure expressed by different organisms

A quimioinformática, definida como o emprego de técnicas informáticas na solução de problemas da química, evolui em conjunto com o desenvolvimento de ferramentas computacionais e é de grande relevância para o planejamento racional de fármacos ao otimizar etapas do desenvolvimento de novas moléculas e economizar recursos e tempo. Dentre as técnicas disponíveis destacam-se o planejamento de fármacos baseado na estrutura e no ligante, que quando combinadas auxiliam na identificação e otimização de moléculas ativas frente a alvos farmacológicos. A Dihidrofolato Redutase (DHFR) é uma importante enzima da via dos folatos que catalisa a redução do dihidrofolato em tetrahidrofolato, utilizando NADPH como cofator, reação essencial para a replicação celular, visto que este ciclo resulta na síntese de precursores das bases nitrogenadas que compõem o DNA, consequentemente, inibidores de DHFR são utilizados no tratamento de infecções bacterianas e alguns tipos de câncer. Trypanosoma cruzi, protozoário causador da doença de chagas, é um dos organismos que expressam a DHFR, além do próprio Homo sapiens. Analisaram-se ligantes conhecidos e as estruturas da proteína expressa pelos dois organismos, visando identificar pontos de divergência que possam ser explorados no planejamento de moléculas seletivas para o tratamento da doença de Chagas. Os 6 modelos cristalográficos de T. cruzi e 2 de H. sapiens foram obtidos do banco de dados de proteínas (PDB) após aplicação de filtros de qualidade. Foram analisadas as sequências de aminoácidos dos modelos, com o uso do Cluster Ômega, sua estrutura tridimensional com os programas Pymol e Chimera X, além da análise das cavidades proteicas com o CavityPlus, que também gerou os farmacóforos de ambos alvos. A análise de estrutura primária identificou mutações em três aminoácidos nos cristais do parasita, que podem ser explicados por diferentes caminhos evolutivos de grupos segregados, embora nenhuma mutação observada esteja em regiões de sítio ativo. A análise dos modelos permitiu que fossem identificados os 25 aminoácidos que estão a menos de 5 Å de distância dos ligantes de T. cruzi, sendo 5 aminoácidos responsáveis por interações de hidrogênio com pelo menos um dos ligantes analisados. Destes, 18 se repetem na proteína humana ou são substituídos por outro aminoácido que mantém a mesma interação. Quanto às diferenças observadas, destacam-se a asparagina 44 substituída por uma prolina na proteína humana e a prolina 92, substituída por uma lisina. A análise de cavidades identificou três cavidades em cada proteína, embora somente as cavidades correspondentes ao sítio ativo sejam druggables. A cavidade da proteína humana é maior e mais alongada, além de apresentar o aspecto de um túnel, enquanto a cavidade da proteína parasita é mais aberta, tal abertura permite que ligantes com o anel benzeno meta substituídos explorem uma região existente na cavidade de T. cruzi que é fechada na humana. O farmacóforo de ambas proteínas foi identificado, apresentando diferenças no tamanho e angulação que também podem ser explorados no planejamento de fármacos seletivos

Chemoinformatic, defined as the use of informatic techniques to solve chemical problems, has evolved together with new computational tools and it is quite important for rational drug designing, by optimizing different steps on the development pipeline of new molecules, saving resources and time. From all the available tools, structure and ligand based drug design shall be highlighted, when combined, they support the identification and optimization of active molecules from pharmaceutical targets. Dihydrofolate reductase (DHFR) is an important enzyme of the folate pathway that catalyzes the reduction of dihydrofolate to tetrahydrofolate, by using NADPH as cofactor. This reaction is essential for cell replication, as this pathway results in the synthesis of nucleobases that build the DNA. That's the reason why DHFR inhibitors are used for treating bacterial infections and some types of cancer. Trypanosoma cruzi, a protozoa that causes Chagas disease, is one of the organisms that express DHFR, besides Homo sapiens itself. This work analyzed known ligands and the structure of the protein expressed by both organisms, aiming to identify divergence points that could be explored for designing selective drugs for Chagas disease treatment. The 6 proteins crystallographic models from T. cruzi and 2 from H. sapiens were obtained from protein data bank (PDB) after the application of quality filters. The amino acid sequence of each model was analyzed by Clustal Omega, its tridimensional structure by Pymol and Chimera X and the cavity analysis by CavityPlus, that also generated the pharmacophore from both targets. The primary structure analysis identified mutations on three amino acids on the parasite christal, which may be explained by different evolutive paths from segregated groups, although none of the observed mutations are on the active site region. The model's analysis allowed the identification of 24 amino acids that are closer than 5 Å from the T. cruzi ligands, 5 of them responsible for hydrogen interactions on at least one of the ligands analyzed. 18 of them are repeated on the human protein or are replaced by another amino acid that preserves the same interaction. As by the differences observed that shall be highlighted, asparagine 44 is replaced by a proline on the human protein, and proline 92 by a lysin. The cavity analysis identified three cavities on each protein, although only the cavities of the active site are druggables. The human protein cavity is bigger and longer, besides it looks like its a tunnel, when the parasite protein is open, that opening allows ligands with benzene ring meta substituted to explore the existing regions of the T. cruzi protein that is closed on the human protein. Lastly, the pharmacophore from both proteins was identified, it shows differences on size and angulation that also could be explored in the designing of selective drugs

In Silico Design of a Chimeric Humanized L-asparaginase.

Acute lymphoblastic leukemia (ALL) is the most common cancer among children worldwide, characterized by an overproduction of undifferentiated lymphoblasts in the bone marrow. The treatment of choice for this disease is the enzyme L-asparaginase (ASNase) from bacterial sources. ASNase hydrolyzes circulating L-asparagine in plasma, leading to starvation of leukemic cells. The ASNase formulations of E. coli and E. chrysanthemi present notorious adverse effects, especially the immunogenicity they generate, which undermine both their effectiveness as drugs and patient safety. In this study, we developed a humanized chimeric enzyme from E. coli L-asparaginase which would reduce the immunological problems associated with current L-asparaginase therapy. For these, the immunogenic epitopes of E. coli L-asparaginase (PDB: 3ECA) were determined and replaced with those of the less immunogenic Homo sapiens asparaginase (PDB:4O0H). The structures were modeled using the Pymol software and the chimeric enzyme was modeled using the SWISS-MODEL service. A humanized chimeric enzyme with four subunits similar to the template structure was obtained, and the presence of asparaginase enzymatic activity was predicted by protein-ligand docking.

Identifying the origin of acrylamide in Peruvian panela production to inform strategies for its reduction.

Maximum levels of acrylamide have been set by the European Commission (EU) 2017/2158 for several food products due to its carcinogenic properties. Although not regulated yet, European buyers are requesting maximum levels of 0.8 mg kg-1 in artisanal panela (raw cane sugar) from northern Peru. Panela in this area is produced by 600 small holder farmers and exportation guarantees a respectable price in an area with a high index of poverty. The objective here was to determine the cause of high acrylamide concentrations in panela to inform cost effective minimisation strategies. We monitored panela production from field to final product to understand the scale of the problem, identify the cause of acrylamide formation, as well as the effect of storage on its concentration. We also determined the utility of rapid kits for asparagine quantification. Our results indicate that high acrylamide levels are a widespread problem (85% of samples analysed) and there was a correlation between acrylamide and asparagine of R2 = 0.58 (p < 0.001), but not with any post-harvest processing variable. We estimate that with a concentration of asparagine of <0.1 g l-1 in sugarcane juice, the threshold set by buyers for acrylamide can be met. Potential solutions to reduce asparagine include varietal selection, improved agronomic practices and the use of asparaginase during panela production. However, any proposed measure should be applicable in the context of the rural Peru. Additionally, we confirm the utility of rapid and low-cost kits for measuring asparagine. This pioneering study provides a baseline for effective management for acrylamide minimization in panela.

A new extracellular glutaminase and urease-free L-asparaginase from Meyerozyma guilliermondii.

L-Asparaginase (L-ASNase) is a potent chemotherapeutic drug employed to treat leukemia and lymphoma. Currently, L-ASNases for therapeutic use are obtained from Escherichia coli and Dickeya chrysanthemi (Erwinia chrysanthemi). Despite their therapeutic potential, enzymes from bacteria are subject to inducing immune responses, resulting in a higher number of side effects. Eukaryote producers, such as fungi, may provide therapeutic alternatives through enzymes that induce relatively less toxicity and immune responses. Additional expected benefits from yeast-derived enzymes include higher activity and stability in physiological conditions. This work describes the new potential therapeutic candidate L-ASNase from the yeast Meyerozyma guilliermondii. A statistical approach (full factorial central composite design) was used to optimize L-ASNase production, considering L-asparagine and glucose concentration, pH of the medium, and cultivation time as independent factors. In addition, the crude enzymes were biochemically characterized, in terms of temperature and optimal pH, thermostability, pH stability, and associated glutaminase or urease activities. Our results showed that enzyme production increased after supplementing a pH 4.0 medium with 1.0% L-asparagine and 0.5% glucose during 75 h of cultivation. Under these optimized conditions, L-ASNase production reached 26.01 U mL-1, which is suitable for scale-up studies. The produced L-ASNase exhibits maximal activity at 37 °C and pH 7.0 and is highly stable under physiological conditions. In addition, M. guilliermondii L-ASNase has no associated glutaminase or urease activities, demonstrating its potential as a promising antineoplastic agent.

Elucidation of the Reaction Mechanism of Cavia porcellus l-Asparaginase: A QM/MM Study.

The l-asparaginase (l-ASNase) enzyme catalyzes the conversion of the non-essential amino acid l-asparagine into l-aspartic acid and ammonia. Importantly, the l-ASNases are used as a key part of the treatment of acute lymphoblastic leukemia (ALL); however, despite their benefits, they trigger severe side effects because they have their origin in bacterial species (Escherichia coli and Erwinia chrysanthemi). Therefore, one way to solve these side effects is the use of l-ASNases with characteristics similar to those of bacterial types, but from different sources. In this sense, Cavia porcellus l-ASNase (CpA) of mammalian origin is a promising enzyme because it possesses similarities with bacterial species. In this work, the hydrolysis reaction for C. porcellus l-asparaginase was studied from an atomistic point of view. The QM/MM methodology was employed to describe the reaction, from which it was found that the conversion mechanism of l-asparagine into l-aspartic acid occurs in four steps. It was identified that the nucleophilic attack and release of the ammonia group is the rate-limiting step of the reaction. In this step, the nucleophile (Thr19) attacks the substrate (ASN) leading to the formation of a covalent intermediate and release of the leaving group (ammonia). The calculated energy barrier is 18.9 kcal mol-1, at the M06-2X+D3(0)/6-311+G(2d,2p)//CHARMM36 level of theory, which is in agreement with the kinetic data available in the literature, 15.9 kcal mol-1 (derived from the kcat value of 38.6 s-1). These catalytic aspects will hopefully pave the way toward enhanced forms of CpA. Finally, our work emphasizes that computational calculations may enhance the rational design of mutations to improve the catalytic properties of the CpA enzyme.

Aliivibrio fischeri L-Asparaginase production by engineered Bacillus subtilis: a potential new biopharmaceutical.

L-Asparaginase (L-ASNase) is an enzyme applied in the treatment of lymphoid malignancies. However, an innovative L-ASNase with high yield and lower side effects than the commercially available preparations are still a market requirement. Here, a new-engineered Bacillus subtilis strain was evaluated for Aliivibrio fischeri L-ASNase II production, being the bioprocess development and the enzyme characterization studied. The pBS0E plasmid replicative in Bacillus sp and containing PxylA promoter inducible by xylose and its repressive molecule sequence (XylR) was used for the genetic modification. Initially, cultivations were carried out in orbital shaker, and then the process was scaled up to stirred tank bioreactor (STB). After the bioprocess, the cells were recovered and submitted to ultrasound sonication for cells disruption and intracellular enzyme recovery. The enzymatic extract was characterized to assess its biochemical, kinetic and thermal properties using L-Asparagine and L-Glutamine as substrates. The results indicated the potential enzyme production in STB achieving L-ASNase activity up to 1.539 U mL-1. The enzymatic extract showed an optimum pH of 7.5, high L-Asparagine affinity (Km = 1.2275 mmol L-1) and low L-Glutaminase activity (0.568-0.738 U mL-1). In addition, thermal inactivation was analyzed by two different Kinect models to elucidate inactivation mechanisms, low kinetic thermal inactivation constants for 25 ºC and 37 ºC (0.128 and 0.148 h-1, respectively) indicate an elevated stability. The findings herein show that the produced recombinant L-ASNase has potential to be applied for pharmaceutical purposes.

Desenvolvimento e caracterização de lipossomas peguilados para veiculação de L-asparaginase / Development and characterization of peg-grafted liposomes for lasparaginase encapsulation

A Leucemia Linfoide Aguda (LLA) é um câncer de maior incidência em crianças, e tem a Lasparaginase (ASNase) como fármaco amplamente utilizado no tratamento dos afetados. A ASNase catalisa a hidrólise do aminoácido L-asparagina (Asn), presente na corrente sanguínea, a ausência do aminoácido no meio extracelular leva à morte células leucêmicas, que necessitam deste aminoácido para as funções celulares. Fatores envolvendo a eficiência do tratamento com ASNase como reações adversas e curta meia-vida, principalmente devido ao reconhecimento pelo sistema imune e degradação por proteases, limitam a sua eficácia. A encapsulação da enzima em lipossomas pode conferir proteção à degradação, melhorar seu perfil farmacocinético e diminuir os efeitos adversos, de forma a melhorar o tratamento da LLA sendo este o objetivo desse trabalho. Lipossomas de DOPC (1,2-dioleoil-sn-glicero-3-fosfocolina) e DMPC (1,2-dimiristoil-snglicero-3-fosfocolina) foram desenvolvidos empregando-se o método de hidratação do filme lipídico e diferentes protocolos de preparo contendo ou não diferentes concentrações de 18:0 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polietilenogicol)-2000] (DSPE-PEG). Os lipossomas produzidos foram utilizados para encapsular a ASNase e os sistemas contendo ou não ASNase encapsulada foram caracterizados por espalhamento de luz dinâmico (DLS), potencial zeta, microscopia eletrônica de transmissão (MET) e criomicroscopia de transmissão. Adicionalmente, foram avaliados a taxa de encapsulação e o perfil de permeabilidade das vesículas à L-asparagina. As análises de DLS mostraram que as nanoestruturas formadas empregando-se agitação magnética a partir de sistemas contendo 10% e 20% de DSPE-PEG possuem diâmetro hidrodinâmico menor (~ 25 nm a 60 nm) que os mesmos sistemas sem o fosfolipídio peguilado (~190 nm a 222 nm), demonstrando a relação entre a diminuição do tamanho e o aumento da quantidade de fosfolipídio peguilado e possível formação de estruturas micelares ou bicelares. O emprego de agitação em vórtex para hidratação do filme lipídico, adição do antioxidante -tocoferol e redução da concentração de DSPE-PEG (5% e 10%) levou à formação de sistemas com diâmetro hidrodinâmico maior, sendo esse protocolo e concentrações de PEG definidos como padrão. As análises de MET comprovaram a formação de lipossomas com diâmetro hidrodinâmico semelhante ao observado por DLS; com a utilização da criomicroscopia foi possível observar os lipossomas sem deformações. Os lipossomas de DMPC/DSPE-PEG 10% apresentaram maior permeabilidade à L-asparagina ao longo do tempo e, portanto, poderiam funcionar como nanoreatores, depletando o aminoácido da circulação. Estudos in vitro com células tumorais devem ser realizados e em seguida estudos in vivo, para confirmar este potencial

L-asparaginase (ASNase) is a first-choice drug, combined with other drugs, in therapeutic schemes to treat Acute Lymphoblastic Leukemia (ALL) in children and adolescents. ASNase catalyzes the hydrolysis of L-asparagine (Asn) in the bloodstream; since ALL cells cannot synthesize this amino acid, protein synthesis is impaired leading to leukemic cells death by apoptosis. In spite of its therapeutic importance, treatment with ASNase is associated to side effects, mainly hypersensitivity and immunogenicity. Another drawback refers to degradation by plasma proteases that altogether with immunogenicity shortens the enzyme half-life. Encapsulation of ASNase in liposomes, vesicular nanostructures formed by the self-aggregation of phospholipids, is an attractive alternative that possibly will protect the enzyme from plasma proteases, resulting on better pharmacokinetics profile. In this work, we prepared by thin film hydration liposomal formulations of the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC) containing or not different concentrations of 18:0 1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG), and encapsulated ASNase by electroporation. The systems containing or not ASNase were analyzed by Dynamic Light Scattering, zeta potential and Electron Microscopy. The encapsulation efficiency and vesicles permeability were also evaluated. According to the DLS analysis, the nanostructures formed by film hydration under magnetic stirring employing 10% or 20% DSPE-PEG presented smaller hydrodynamic diameter (~ 25 nm to 60 nm) than the same systems without the pegylated phospholipid (~ 190 nm to 222 nm), demonstrating the relation between size and the amount of pegylated phospholipid that results in formation of micellar or bicellar structures. The protocol was stabilize by hydration of the lipid film under vortex agitation, addition of the antioxidant - tocopherol and reduction of the concentration of DSPE-PEG (5% and 10%), what altogether led to the formation of nanostructures of higher hydrodynamic diameter and monodisperse systems. TEM analyzes confirmed the formation of liposomes with hydrodynamic diameter similar to that observed by DLS; with the use of cryomicroscopy it was possible to observe the liposomes without deformations. Liposomes of DMPC/DSPE-PEG 10% showed permeability to L-asparagine over time and, therefore, could function as nanoreactors, depleting the circulating amino acid

Technological Prospecting: Mapping Patents on L-asparaginases from Extremophilic Microorganisms.

BACKGROUND: L-asparaginase (L-ASNase, L-asparagine amidohydrolase, E.C.3.5.1.1) is an enzyme with wide therapeutic applicability. Currently, the commercialized L-ASNase comes from mesophilic organisms, presenting low specificity to the substrate and limitations regarding thermostability and active pH range. Such factors prevent the maximum performance of the enzyme in different applications. Therefore, extremophilic organisms may represent important candidates for obtaining amidohydrolases with particular characteristics desired by the biotechnological market. OBJECTIVES: The present study aims to carry out a technological prospecting of patents related to the L-asparaginases derived from extremophilic organisms, contributing to pave the way for further rational investigation and application of such enzymes. METHODS: This patent literature review used six patents databases: The LENS, WIPO, EPO, USPTO, Patent Inspiration, and INPI. RESULTS: It was analyzed 2860 patents, and 14 were selected according to combinations of descriptors and study criteria. Approximately 57.14% of the patents refer to enzymes obtained from archaea, especially from the speciesPyrococcus yayanosii (35.71% of the totality). CONCLUSION: The present prospective study has singular relevance since there are no recent patent reviews for L-asparaginases, especially produced by extremophilic microorganisms. Although such enzymes have well-defined applications, corroborated by the patents compiled in this review, the most recent studies allude to new uses, such as the treatment of infections. The characterization of the catalytic profiles allows us to infer that there are potential sources still unexplored. Hence, the search for new L-ASNases with different characteristics will continue to grow in the coming years and, possibly, ramifications of the technological routes will be witnessed.

A Patent Review on the Use of L-Asparaginase in the Treatment of Acute Lymphocytic Leukemia.

L-asparaginase (L-asparagine amino hydrolase, E.C.3.5.1.1) is the most important chemotherapeutic drug used in treating Acute Lymphocytic Leukemia (ALL), decreasing blood asparagine rates causing apoptosis in tumor cells. However, pharmacological drugs cause several side effects making treatments difficult. Thus, searches for new sources of L-asparaginase or enzyme modifications focus on discovering new products to use in therapy. This article reviewed published patents from 2000 to 2020 related to the treatment of ALL using L-asparaginase. Many organisms have been shown as potential viable L-asparaginase producers for use in the treatment of ALL. However, this patent review shows that few of these organisms are gaining attention to becoming bioproducts for the market. It is expected that drugs in the testing phase and patents related to the treatment of ALL and other cancers will become real products. Besides, a treatment using an amino acid depletion approach, now referring to asparagine, altogether with a compound that directly interferes with the expression of the asparagine synthase gene, is more suitable for the treatment of ALL and possibly to other cancers.

Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins.

Here, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC-MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

Sex/Gender Modifies the Association Between the MC4R p.Ile269Asn Mutation and Type 2 Diabetes in the Mexican Population.

CONTEXT: Studies in mice and humans suggest that melanocortin-4 receptor (MC4R) deficiency affects body weight in a sex-/gender-dependent manner. However, similar evidence for type 2 diabetes (T2D) is scarce. OBJECTIVE AND DESIGN: We investigated whether sex/gender modifies the association between the loss-of-function MC4R p.Ile269Asn mutation and T2D in 6929 Mexican adults (3175 T2D cases and 3754 normal glucose tolerance [NGT] controls). The 2003 American Diabetes Association criteria were used to define NGT and T2D. The MC4R p.Ile269Asn mutation was genotyped in all participants using TaqMan technology. RESULTS: The MC4R p.Ile269Asn mutation was associated with T2D in 6929 Mexican adults (Ncontrols = 3754, Ncases = 3175, odds ratio [OR] = 2.00, 95% confidence interval [CI], 1.35-2.97; P = 5.7 × 10-4). The MC4R p.Ile269Asn mutation had a frequency of 0.86 and 1.05% in women with NGT and T2D, and 0.78 and 1.32% in men with NGT and T2D, respectively. We identified a significant interaction between the MC4R p.Ile269Asn mutation and sex/gender on T2D risk (P = 0.049). Although a strong association between the mutation and T2D was observed in men (Ncontrols = 2418, Ncases = 1807, OR = 2.63, 95% CI, 1.62-4.28, P = 9.3 × 10-5), results were not significant in women (Ncontrols = 1336, Ncases = 1368, OR = 1.16, 95% CI, 0.60-2.26, P = 0.65). Further adjustment for body mass index in the logistic regression model did not alter the sex-/gender-specific pattern of association (men: OR = 2.22, 95% CI, 1.34-3.67, P = 0.0019; women: OR = 1.02, 95% CI, 0.51-2.02, P = 0.95). CONCLUSION: This is the first report of a male-specific association between the MC4R p.Ile269Asn loss-of-function mutation and T2D in the Mexican population.

[New variant in the ALG13 gene responsible for the congenital disorder of Is-type glycosylation in a male patient]. / Nueva variante en el gen ALG13 responsable del trastorno congénito de la glicosilación tipo Is en un paciente masculino.

INTRODUCTION: Congenital disorders of glycosylation (CDGs) are a group of inborn errors of glycan metabolism with multi-systemic manifestations. More than 100 different types of CDGs have been reported. The form involving the asparagine-linked glycosylation 13 (ALG13) gene is an uncommon X-linked form of these pathologies. OBJECTIVE: To describe the clinical features in one patient with ALG13-CDG and to compare them with previously reported cases. CLINICAL CASE: A 11-years-old boy, child of consangui neous parents, with hypotonia, severe developmental delay, intellectual disability, feeding difficulties, congenital heart disease (patent ductus arteriosus and mitral regurgitation), without epilepsy or coa gulation disorders. The metabolic screening showed unclear results, including N-glycosylation stu dies in plasma that were normal. Therefore, whole-exome sequencing (WES) was performed which identified a previously unreported variant in the ALG13 gene: c.428C > T (p.P143L) in hemizygous state; confirmed by Sanger sequencing. His mother was a carrier of the same variant. CONCLUSION: This is the first report of a Colombian patient with ALG13-CDG without epilepsy. The findings in this patient broaden the phenotypic spectrum of ALG13-CDG known to date and support that N- glycosylation disorders may be present in normal biochemical analysis. WES has become a cost- effective technique that allows the identification of disease-causing mutations in diseases with a broad phenotypic and genotypic spectrum.

Activation of regulator ArcA in the presence of hypochlorite in Salmonella enterica serovar Typhimurium.

Oxidative stress is the main mechanism behind efficient disinfectants, causing damage in bacterial macromolecules. Importantly, bacteria activate resistance mechanisms in response to damage generated by oxidative stress. Strategies allowing pathogens to survive oxidative stress are highly conserved among microorganisms. Many of these strategies entail genomic responses triggered by signals transduced through Two Component Systems (TCS). Recently, we demonstrated that the TCS ArcAB (specifically ArcA) participates in bacterial responses to hypochlorite, regulating the uptake of this toxic compound and being involved in resistance and survival inside neutrophils, where hypochlorous acid abounds. Here, we demonstrated that ArcA is required in the response to oxidative stress generated by hypochlorite, independent of its cognate sensor ArcB or the Asp54 of ArcA, the only phosphorylable residue in ArcA, which is required to function as a gene regulator. Our results suggest that ArcA could have additional functions to respond to oxidative stress, independent of its regulatory activity, which might require interaction with other unknown relevant proteins.

Production, purification and characterization of recombinant human R-spondin1 (RSPO1) protein stably expressed in human HEK293 cells.

BACKGROUND: The R-Spondin proteins comprise a family of secreted proteins, known for their important roles in cell proliferation, differentiation and death, by inducing the Wnt pathway. Several studies have demonstrated the importance of RSPOs in regulation of a number of tissue-specific processes, namely: bone formation, skeletal muscle tissue development, proliferation of pancreatic ß-cells and intestinal stem cells and even cancer. RSPO1 stands out among RSPOs molecules with respect to its potential therapeutic use, especially in the Regenerative Medicine field, due to its mitogenic activity in stem cells. Here, we generated a recombinant human RSPO1 (rhRSPO1) using the HEK293 cell line, obtaining a purified, characterized and biologically active protein product to be used in Cell Therapy. The hRSPO1 coding sequence was synthesized and subcloned into a mammalian cell expression vector. HEK293 cells were stably co-transfected with the recombinant expression vector containing the hRSPO1 coding sequence and a hygromycin resistance plasmid, selected for hygror and subjected to cell clones isolation. RESULTS: rhRSPO1 was obtained, in the absence of serum, from culture supernatants of transfected HEK293 cells and purified using a novel purification strategy, involving two sequential chromatographic steps, namely: heparin affinity chromatography, followed by a molecular exclusion chromatography, designed to yield a high purity product. The purified protein was characterized by Western blotting, mass spectrometry and in vitro (C2C12 cells) and in vivo (BALB/c mice) biological activity assays, confirming the structural integrity and biological efficacy of this human cell expression system. Furthermore, rhRSPO1 glycosylation analysis allowed us to describe, for the first time, the glycan composition of this oligosaccharide chain, confirming the presence of an N-glycosylation in residue Asn137 of the polypeptide chain, as previously described. In addition, this analysis revealing the presence of glycan structures such as terminal sialic acid, N-acetylglucosamine and/or galactose. CONCLUSION: Therefore, a stable platform for the production and purification of recombinant hRSPO1 from HEK293 cells was generated, leading to the production of a purified, fully characterized and biologically active protein product to be applied in Tissue Engineering.

Interferences that impact measuring optimal L-asparaginase activity and consequent errors interpreting these data.

L-asparaginase is an enzyme produced by microorganisms, plants, and animals, which is used clinically for the treatment for acute lymphoblastic leukemia (ALL) and, in the food industry, to control acrylamide formation in baked foods. The purpose of this review was to evaluate the available literature regarding microbial sources of L-asparaginase, culture media used to achieve maximum enzyme expression in microbial fermentations, and assay methods employed to assess L-asparaginase activity. Studies were gathered by searching PubMed, and Web of Science databases before January 22, 2018, with no time restrictions. The articles were evaluated according to the source of L-asparaginase being studied, the nitrogen source in the culture medium, the type of sample, and the method employed to evaluate L-asparaginase activity. Bacterial L-asparaginase appeared to be the most commonly studied source of the enzyme and, most often, the enzyme activity was assayed from crude protein extracts using the Nessler method, which is an indirect measurement of asparaginase activity that determines the concentration of ammonia generated after the action of the enzyme on the substrate, L-asparagine. However, ammonia is also generated throughout microbial fermentations and this endogenous ammonia will also reduce the Nessler reagent if crude microbial extracts are used to determine total L-asparaginase activity. We suggest that current estimates of L-asparaginase activity reported in the literature may be overestimated when Nessler reagent is used, since we were unable to find a single study that made reference to the possible inference of fermentation derived ammonia.

Influence of functional variants Asp312Asn and Lys751Gln of Xeroderma Pigmentosum Group D (XPD) and Glutathione S-transferase Mu 1 (GSTM1) and Theta 1 (GSTT1) genes on cutaneous melanoma susceptibility and prognosis.

We aimed to evaluate whether variants in repair (XPD Asp312Asn, XPD Lys751Gln) and detoxification (GSTM1, GSTT1) genes alter risk, clinicopathological aspects and survival of cutaneous melanoma (CM). Genotyping was performed in 229 CM patients and 258 controls. Individuals with XPD 312Asp/Asn or Asn/Asn plus GSTT1 null genotype were under 2.00 (95% CI: 1.06-3.79), and XPD 312Asn/Gln haplotype was under 1.44-fold (95% CI: 0.99-2.08) increased risks to CM than others. Individuals with GSTM1 plus GSTT1 null genotype had 9.61-fold (95% CI: 2.28-40.38) increased risk of metastatic CM. At 60 months of follow-up, patients with XPD 751Gln/Gln plus GSTT1 null and GSTM1 null plus GSTT1 null genotype presented 7.36 and 3.05 more chances of evolving to death in multivariate Cox analysis, respectively. In conclusion, our data indicate, for the first time, that specific variant combinations of XPD, GSTM1 and GSTT1 may increase susceptibility to CM and influence patients' clinicopathological features and survival.

Expression, purification, and characterization of asparaginase II from Saccharomyces cerevisiae in Escherichia coli.

l-asparaginase catalyzes the conversion of l-asparagine to l-aspartate and ammonium. This protein is an important therapeutic enzyme used for the treatment of acute lymphoblastic leukemia. In this study, the asparaginase II-encoding gene ASP3 from Saccharomyces cerevisiae was cloned into the expression vector pET28a in-fusion with a 6x histidine tag and was expressed in Escherichia coli BL21 (DE3) cells. The protein was expressed at a high level (225.6 IU/g cells) as an intracellular and soluble molecule and was purified from the supernatant by nickel affinity chromatography. The enzyme showed very low activity against l-glutamine. The denaturing electrophoresis analysis indicated that the recombinant protein had a molecular mass of ∼38 kDa. The native enzyme was a tetramer with a molecular mass of approximately 178 kDa. The enzyme preparation showed antitumor activity against the K562 and Jurkat cell lines comparable or even superior to the E. coli commercial asparaginase.