Production of a novel N-terminal PEGylated crisantaspase.

Crisantaspase is an asparaginase enzyme produced by Erwinia chrysanthemi and used to treat acute lymphoblastic leukemia (ALL) in case of hypersensitivity to Escherichia coli l-asparaginase (ASNase). The main disadvantages of crisantaspase are the short half-life (10 H) and immunogenicity. In this sense, its PEGylated form (PEG-crisantaspase) could not only reduce immunogenicity but also improve plasma half-life. In this work, we developed a process to obtain a site-specific N-terminal PEGylated crisantaspase (PEG-crisantaspase). Crisantaspase was recombinantly expressed in E. coli BL21(DE3) strain cultivated in a shaker and in a 2-L bioreactor. Volumetric productivity in bioreactor increased 37% compared to shaker conditions (460 and 335 U L-1  H-1 , respectively). Crisantaspase was extracted by osmotic shock and purified by cation exchange chromatography, presenting specific activity of 694 U mg-1 , 21.7 purification fold, and yield of 69%. Purified crisantaspase was PEGylated with 10 kDa methoxy polyethylene glycol-N-hydroxysuccinimidyl (mPEG-NHS) at different pH values (6.5-9.0). The highest N-terminal pegylation yield (50%) was at pH 7.5 with the lowest poly-PEGylation ratio (7%). PEG-crisantaspase was purified by size exclusion chromatography and presented a KM value three times higher than crisantaspase (150 and 48.5 µM, respectively). Nonetheless, PEG-crisantaspase was found to be more stable at high temperatures and over longer periods of time. In 2 weeks, crisantaspase lost 93% of its specific activity, whereas PEG-crisantaspase was stable for 20 days. Therefore, the novel PEG-crisantaspase enzyme represents a promising biobetter alternative for the treatment of ALL.

Influence and effect of osmolytes in biopharmaceutical formulations.

Osmolytes are small organic molecules accumulated by cells in response to environmental stresses. They are represented by amino acids, sugars, polyols, tertiary sulphonium and quaternary ammonium compounds. These molecules present a protective behaviour and favour the equilibrium of macromolecules towards the native form, preventing denaturation and promoting the folding of unfolded proteins. Protein formulations due to their biological character require greater care during the manufacturing process, shelf-life and administration of the drug, as variations in these factors may result in denaturation, inactivation and/or protein aggregation. These drawbacks can be surpassed using osmolytes as excipients in protein formulations as stabilisers, bulking agents and even buffers. A number of 133 biologics, including vaccines and immunoglobulins, approved by the U.S. Food and Drug Administration (FDA) between 1998 and 2017 were analysed in this work in order to identify the most used group of osmolyte molecules. A deep insight into their role in protein formulations was discussed and compared to data in the literature. The advantages and disadvantages of their use in specific formulations were also extensively discussed here. In conclusion, investigation into the role of osmolytes in each formulation is essential for understanding their effect and provides a background to be used when selecting the best osmolyte to fit a specific formulation without excluding the patient needs.

Caracterização de L-Asparaginase de Erwinia chrysanthemi melhorada por evolução sintética de proteí­nas e otimização das condições de produção / Characterization of Erwinia chrysanthemi L-Asparaginase improved by synthetic protein evolution and optimization of production conditions

A L-Asparaginase (L-ASNase) é uma enzima tetramérica bacteriana, utilizada em sessões de quimioterapia. Essa enzima depleta os aminoácidos asparagina (Asn) e glutamina (Gln), transformando-os em aspartato (Asp) ou glutamato (Glu), respectivamente, e em amônia. Contudo, a L-ASNase pode induzir resposta imune, levando à produção de anticorpos antiasparaginase, uma causa importante de resistência ao medicamento. Uma L-ASNase ideal seria aquela com alta atividade e estabilidade e baixo potencial imunogênico, porém, as L-ASNases utilizadas na terapêutica não reúnem essas características simultaneamente. Por essa razão, o presente trabalho utilizou técnicas de mutagênese randômica, a fim de criar uma nova proteoforma de L-ASNase de E. chrysanthemi com uma melhor atividade e estabilidade. Além disso, foram estudadas condições de cultivo em agitador metabólico, visando à otimização de condições de produção. Foi criada uma biblioteca com 1.056 clones, e desses, 19 foram selecionados por apresentarem atividade superior ou igual à enzima selvagem quando dosada em extrato bruto. Dentre eles, dois mutantes se destacaram por apresentarem a atividade específica glutaminásica diferente da enzima selvagem. Análises in silico indicam que o mutante 9-6D apresentou diminuição de desordem estrutural e epítopos imunogênicos. O mutante 9-5F demonstrou uma diminuição da porcentagem da atividade glutaminásica quando comparada a enzima selvagem. O estudo de produção do mutante 9-5F indicou que a temperatura de indução, seguida da concentração do indutor, são os parâmetros mais relevantes para a otimização da produção de L-ASNase de E. chrysanthemi mutante

L-Asparaginase (L-ASNase) is a bacterial tetrameric enzyme used in chemotherapy sessions that deplete asparagine (Asn) and glutamine (Gln), transforming them into Aspartate (Asp) or glutamate (Glu), respectively, and ammonia. However, L-ASNase can induce immune response leading to the production of anti-asparaginase antibody, an important cause of drug resistance. Ideally, L-ASNase would be one with high activity, high stability and low immunogenic potential, but the L-ASNases commercially available today do not present these characteristics simultaneously. For this reason, this study used techniques of random and site-directed mutagenesis in order to create a new proteoform of E. chrysanthemi L-ASNase with improved activity and stability. In addition, culture conditions were studied in a metabolic shaker, aiming at the optimization of production conditions. A library with 1,056 clones was created, and of these clones, 19 were selected because they had activity superior or equal to the wild-type enzyme in crude protein extract. Among them, 2 mutants stood out for having different glutaminase specific activity in relation to wild-type enzyme. The 9-6D mutant also showed decreased structural disorder and immunogenic epitopes. The 9-5F mutant demonstrated a decrease in percentage of glutaminase activity when compared to the wild-type enzyme. The production study of 9-5F mutant indicated that the induction temperature followed by the inductor concentration are the most relevant parameters for the production optimization of E. chrysanthemi mutant L-ASNase

Chemogenomic Study of Carboplatin in Saccharomyces cerevisiae: Inhibition of the NEDDylation Process Overcomes Cellular Resistance Mediated by HuR and Cullin Proteins.

The use of carboplatin in cancer chemotherapy is limited by the emergence of drug resistance. To understand the molecular basis for this resistance, a chemogenomic screen was performed in 53 yeast mutants that had previously presented strong sensitivity to this widely used anticancer agent. Thirty-four mutants were responsive to carboplatin, and from these, 21 genes were selected for further studies because they have human homologues. Sixty percent of these yeast genes possessed human homologues which encoded proteins that interact with cullin scaffolds of ubiquitin ligases, or whose mRNA are under the regulation of Human antigen R (HuR) protein. Both HuR and cullin proteins are regulated through NEDDylation post-translational modification, and so our results indicate that inhibition of this process should sensitise resistant tumour cells to carboplatin. We showed that treatment of a tumour cell line with MLN4924, a NEDDylation inhibitor, overcame the resistance to carboplatin. Our data suggest that inhibition of NEDDylation may be a useful strategy to resensitise tumour cells in patients that have acquired carboplatin resistance.

Alimentación, actividad física y otros factores de riesgo cadiometabólico en la población inmigrante en España. Revisión bibliográfica / Diet, physical activity and other cardiometabolic risk factors in the immigrant population in Spain. a review

Fundamentos: La transición "epidemiológica" de la población inmigrante en el mundo, y en particular en España, es insuficientemente comprendida, tanto porque la morbimortalidad es multicausal como por las limitaciones de información sobre los estilos de vida de los inmigrantes. Por ello, el objetivo de este trabajo fue conocer los factores de riesgo conductuales y biológicos de la enfermedad cardiometabólica en la población inmigrante en España. Métodos: Revisión de conjunto de la literatura publicada durante el periodo 1998-2012. Se seleccionaron artículos en español o inglés, con participantes de Latinoamérica, África, Asia y Europa del Este o que cumplieran la definición de inmigrante de la Organización Internacional de Migraciones. La búsqueda bibliográfica se realizó en Medline y MEDES. Resultados: Se identificaron 117 artículos de los que seleccionaron 16 . 13 de ellos se publicaron desde 2009. 15 fueron estudios transversales y uno un ensayo no randomizado. 5 se realizaron en población general, 7 con muestras de pacientes y 4 en ámbitos mixtos (comunitario y clínico). En 9 estudios el tamaño muestral fue menor de 500 personas y 15 usaron muestras locales o regionales. 13 artículos estudiaron la alimentación y el estado nutricional de los inmigrantes pero hubo mucha heterogeneidad de objetivos y resultados. Algunos estudios mostraban que la frecuencia de obesidad es mayor en población inmigrante que en la originaria de España, que la duración de la residencia en España no se asociaba con la obesidad y que los inmigrantes consumían menos tabaco y alcohol y realizaban menos actividad física que las personas nacidas en España. Conclusiones: La producción científica sobre estilos de vida y factores de riesgo cardiometabólico en inmigrantes en España es reciente y escasa, por lo que no permite conocer suficientemente el perfil de riesgo de esta población (AU)

Background: The "epidemiological transition" of the immigrant population in the world, and particularly in Spain, is insufficiently understood, due to the multi-causality of the morbi-mortality and the limitations of the information about the lifestyles of immigrants. Thus, the objective of this work was to know behavioural and biological risk factors of cardiometabolic disease in the immigrant population in Spain. Methods: Scoping review of the literature published in the period 1998-2012.We selected articles in Spanish or English, with study participants from Latin-America, Africa, Asia and Eastern Europe or who comply with the immigrant definition from the International Organization for Migration. Bibliographic search was performed in Medline and MEDES.. Results: We identified 117 articles, and 16 were included in this review. Thirteen studies were published since 2009. In total, 15 articles corresponded to cross-sectional studies and one to a non-randomized trial; five were population-based, seven were conducted within a clinical setting, and four in mixed settings (population and clinic). In nine studies the sample was less than 500 participants, and 15 studies were conducted at the local or regional level. Thirteen articles focused on food habits and nutritional status, but showed substantial heterogeneity in objectives and results. Some studies found that the frequency of obesity was higher in the immigrant than in the Spanish native population, that the length of residence in Spain was not associated with obesity, and that the immigrants consumed less tobacco and alcohol but did less physical activity than the people born in Spain. Conclusion: The scientific production on the lifestyle and cardiometabolic risk factors among the immigrants in Spain is quite recent and scarce. Thus, it does not allow for characterizing the risk profile of this population (AU)

[Diet, physical activity and other cardiometabolic risk factors in the immigrant population in Spain: a review]. / Alimentación, actividad física y otros factores de riesgo cardiometabólico en la población inmigrante en España: revisión bibliográfica.

BACKGROUND: The "epidemiological transition" of the immigrant population in the world, and particularly in Spain, is insufficiently understood, due to the multi-causality of the morbi-mortality and the limitations of the information about the lifestyles of immigrants. Thus, the objective of this work was to know behavioural and biological risk factors of cardiometabolic disease in the immigrant population in Spain. METHODS: Scoping review of the literature published in the period 1998-2012. We selected articles in Spanish or English, with study participants from Latin-America, Africa, Asia and Eastern Europe or who comply with the immigrant definition from the International Organization for Migration. Bibliographic search was performed in Medline and MEDES. RESULTS: We identified 117 articles, and 16 were included in this review. Thirteen studies were published since 2009. In total, 15 articles corresponded to cross-sectional studies and one to a non-randomized trial; five were population-based, seven were conducted within a clinical setting, and four in mixed settings (population and clinic). In nine studies the sample was less than 500 participants, and 15 studies were conducted at the local or regional level. Thirteen articles focused on food habits and nutritional status, but showed substantial heterogeneity in objectives and results. Some studies found that the frequency of obesity was higher in the immigrant than in the Spanish native population, that the length of residence in Spain was not associated with obesity, and that the immigrants consumed less tobacco and alcohol but did less physical activity than the people born in Spain. CONCLUSION: The scientific production on the lifestyle and cardiometabolic risk factors among the immigrants in Spain is quite recent and scarce. Thus, it does not allow for characterizing the risk profile of this population.