Purification of plasmid DNA targeting therapeutic applications still presents many challenges, namely on supports and specific ligand development. Monolithic supports have emerged as interesting approaches for purifying pDNA due to its excellent mass transfer properties and higher binding capacity values. Moreover, arginine ligands were already described to establish specific and preferential interactions with pDNA. Additionally, some studies revealed the ability of arginine based cationic peptides to condense plasmid DNA, which increased lengthening can result in strongest interactions with higher binding capacities for chromatographic purposes of large molecules such as pDNA. In this work, arginine homopeptides were immobilized in monolithic supports and their performance was evaluated and compared with a single arginine monolithic column regarding supercoiled (sc) plasmid DNA purification. Specific interactions of arginine based peptides with several nucleic acids present in a clarified Escherichia coli lysate sample showed potential for the sc pDNA purification. Effectively, the immobilization of the arginine homopeptides became more functional compared with the single arginine amino acid, showing higher binding capacities, which was also reflected in the intensity of the interactions. The combination of structural versatilities of monoliths with the specificity of arginine peptides raised as a promising strategy for sc pDNA purification.

Frequently, skin is subjected to damaging events, such as deep cuts, burns or ulcers, which may compromise the integrity of this organ. To overcome such lesions, different strategies have been employed. Among them, wound dressings aimed to re-establish skin native properties and decreased patient pain have been pursued for a long time. Herein, an electrospun membrane comprised by deacetylated/arginine modified chitosan (CH-A) was produced to be used as a wound dressing. The obtained results showed that the membrane has a highly hydrophilic and porous three-dimensional nanofibrous network similar to that found in human native extracellular matrix. In vitro data indicate that human fibroblasts adhere and proliferate in contact with membranes, thus corroborating their biocompatibility. This nanofiber-based biomaterial also demonstrated bactericidal activity for two bacterial strains. In vivo application of CH-A nanofibers in full thickness wounds resulted in an improved tissue regeneration and faster wound closure, when compared to non-modified membranes. Such findings support the suitability of using this membrane as a wound dressing in a near future.

The increasing number of applications requiring highly purified plasmid DNA (pDNA) generates a corresponding need for simple, scalable, and cost-effective purification processes. Due to the pDNA large size and complex shape, the use of commercial chromatographic beads often results in poor yields and low binding capacities when operated in a positive mode. An alternative to overcome this limitation is the design of chromatographic ligand-resin systems able to efficiently operate in negative mode, where host impurities (especially low molecular weight RNA) are efficiently captured and separated from the target pDNA. In this work, arginine amino acid and di-arginine peptide (arginine-arginine) were immobilized in agarose resins and evaluated for negative chromatographic purification of pDNA from bacterial cell lysates. The results showed that RNA was preferentially bound to the ligands, interfering with the binding of pDNA. The amount of plasmid processed per column volume by arginine and di-arginine, under negative mode, was substantially larger comparing with the conventional positive mode, resulting in pDNA recoveries up to 99%, with a considerable reduction of host impurities. This study shows that negative mode chromatography using arginine-based ligands poses as an interesting alternative for intermediate and polishing pDNA purification operations, with considerable economic and environmental advantages.

The relevance of RNA in many biological functions has been recognized, broadening the scope of RNA research activities, from basic to applied sciences, also aiming the translation to clinical fields. The preparation and purification of RNA is a critical step for further application, since the quality of the template is crucial to ensure reproducibility and biological relevance. Therefore, the establishment of new tools that allows the isolation of pure RNA with high quality is of particular importance. New chromatographic strategies for RNA purification were considered, exploiting affinity interactions between amino acids and nucleic acids. In the present study, a single arginine-affinity chromatography step was employed for the purification of RNA from a total eukaryotic nucleic acid extract, thus eliminating several steps compared with current RNA isolation procedures. The application of this process resulted in a high RNA recovery yield of 96 ± 17% and the quality control analysis revealed a high integrity (28S:18S ratio = 1.96) in RNA preparations as well as a good purity, demonstrated by the scarce detection of proteins and the reduction on genomic DNA contamination to residual concentrations. Furthermore, the performance of the new RNA isolation method was tested regarding the applicability of the isolated RNA in modern molecular biology techniques. Hence, this new affinity approach will simplify the isolation and purification of RNA, which can bring great improvements in biomedical investigation.

In the search for improved animal performance, has been testing new substances and new products to be used as additives in feed. Creatine and their precursors are examples of such compounds used for improvement of animal productivity. The guanidinoacetic acid is a natural precursor of creatine that has been studied in order to verify their effects on animal performance. With the objective of verify whether supplementation of guanidinoacetic acid in diets could help save the arginine used for the synthesis of creatine, which can be diverted to protein synthesis, growth and development of the animal. 90 piglets weaned at 21 days of age were used. The animals were distributed in a randomized block design based on the initial weight of the animals, with five treatments (0.00, 0.05, 0.10, 0.15 and 0.20% of inclusion of guanidinoacetic acid) six replicates and three animals per experimental unit. The experimental diets and water were fed ad libitum in the pre-initial I phase (21 -32 days old) and pre-initial II (33-42 days old), and the initial phase (43-63 days old). At 42 and 63 days old blood was collected from an animal for experimental unit to analyze creatinine and creatine kinase. They were analyzed daily weight gain, daily feed intake and feed conversion in phases pre-initial (21-42 days old), initial (43-63 days old) and the total duration of the experiment (21-63 days old). All variables were subjected to analysis of variance and regression analysis. It was adopted α = 0.05 and considered trend between α 0,05 and 0,10. In the pre-initial phase (21-42 days), there was a tendency to parameter gain weight daily (P= 0,069), however daily feed intake and feed conversion were not affected significantly by the addition of different levels of guanidinoacetic acid in the diet. As for the blood variables, creatinine levels both at 42 as at 63 days were not significantly affected. For creatine kinase levels at 42 days there was a tendency (P = 0.077), but after 63 days there were significant influences. In the initial phase (43-63 days) and total experimental period (21-63 days) were no significant effects of the inclusion of guanidinoacetic acid, both for performance variables as for creatinine and creatine kinase.

A radioterapia é amplamente utilizada no tratamento de tumores pélvicos, incluindo os de bexiga, intestino e reto. Ela apresenta efeitos danosos, notadamente em tecidos que apresentam intensa renovação celular, sendo a mucosa intestinal altamente susceptível. Nesse contexto, a suplementação dietética com aminoácidos tem se mostrado uma terapia promissora para minimizar este dano. O objetivo desse estudo foi determinar o efeito da suplementação dietética com os aminoácidos L-arginina e glicina na estrutura da parede do cólon de ratos submetidos a irradiação abdominal. Quarenta ratos Wistar machos adultos foram distribuídos aleatoriamente em quatro grupos, cada um com dez animais: I controle não irradiado e sem suplementação de aminoácidos; II controle irradiado e sem suplementação de aminoácidos; III irradiado e suplementado com L-arginina; IV irradiado e suplementado com glicina. O período de suplementação dietética foi de 14 dias, com a irradiação ocorrendo no 8.º dia do experimento. A análise estereológica mostrou que a irradiação provocou diminuição do volume total da parede colônica dos animais dos grupos II e III em relação aos animais saudáveis, mas não dos ratos que receberam suplementação de glicina. A camada mucosa dos animais irradiados de todos os grupos diminuiu quando comparada com os ratos saudáveis não irradiados. Os animais irradiados que não receberam suplementação de aminoácido apresentaram diminuição da camada muscular da mucosa, quando comparados com os grupos I e IV, e o grupo de ratos suplementados com glicina apresentou aumento significativo da camada submucosa em relação aos grupos I e II. Os animais do grupo III mostraram diminuição da camada muscular própria em comparação aos grupos I e IV. A suplementação com L-arginina foi eficaz na manutenção do volume parcial do epitélio da camada mucosa. Nossos resultados sugerem que a suplementação de glicina apresentou efeitos superiores ao da suplementação com L-arginina na estrutura da parede colônica, haja vista que foi capaz de manter a espessura da parede e a superfície epitelial da mucosa, enquanto a Larginina foi capaz de manter o volume parcial do epitélio e a superfície epitelial, mas não o volume total da parede intestinal.

A L-arginina é reconhecida como um nutriente de fundamental importância na resposta imune, apesar de seus efeitos serem, por vezes, considerados inconstantes. O autoimplante esplênico tem sido proposto como alternativa à esplenectomia total isolada, mas existem preocupações quanto à eficácia do restabelecimento da resposta imune, haja vista que o paciente pode permanecer com risco aumentado de desenvolvimento de infecção fulminante pós esplenectomia, mesmo após a regeneração morfológica do órgão. O objetivo deste estudo foi avaliar a participação da suplementação dietética com L-arginina em subpopulações linfocitárias no sangue, no baço e nos autoimplantes esplênicos de ratos submetidos a esplenectomia isolada ou combinada com autoimplante esplênico. Foram utilizados 42 ratos Sprague-Dawley machos, randomicamente distribuídos em seis grupos: 1 Controle operação simulada; 2 esplenectomia total; 3 esplenectomia total combinada com autoimplante esplênico; 4 Controle operação simulada, com suplementação de L-arginina; 5 esplenectomia total, com suplementação de L-arginina; e 6 esplenectomia total combinada com autoimplante esplênico, com suplementação de L-arginina. Os animais dos grupos 4, 5 e 6 receberam suplementação de L-arginina, uma vez ao dia, durante 15 dias anteriores a coleta sangüínea realizada imediatamente antes dos procedimentos operatórios (semanas 0 e 12). A dose utilizada foi de 1,0 g/kg/dia, administrada por via intragástrica em bolus. As avaliações foram realizadas por meio de hemograma e citometria de fluxo. A análise estatística utilizou testes paramétricos e nãoparamétricos, sendo p<0,05 considerado para a rejeição da hipótese nula. A suplementação com L-arginina acarretou elevação da contagem relativa e absoluta de neutrófilos periféricos, 12 semanas após a realização de esplenectomia total combinada com autoimplante esplênico. A esplenectomia total ocasionou diminuição da contagem relativa de linfócitos T totais, T CD4+ e T CD8β no sangue, mas a suplementação dietética com L-arginina evitou a diminuição do percentual de células T totais e T CD8β no sangue dos animais submetidos a autoimplante esplênico. Tanto a realização de autoimplante esplênico como a suplementação de L-arginina previnem a diminuição da subpopulação de linfócitos T CD4+ no sangue periférico, fato que usualmente ocorre após realização de esplenectomia total. Houve maior proliferação de células brancas / g de tecido nos autoimplante esplênico dos animais suplementados, porém a suplementação não influenciou a contagem de linfócitos T, T CD4+ e B de zona marginal de baço. A suplementação do aminoácido L-arginina após a realização de esplenectomia total combinada com autoimplante esplênico em ratos foi capaz de reverter alterações observadas em algumas das subpopulações linfocitárias, ocasionadas pela esplenectomia.

Evidências crescentes têm demonstrado que o exercício prejudica a estrutura da membrana eritrocitária, como consequência do aumento do estresse físico e químico. O óxido nítrico (NO) derivado dos eritrócitos afeta a fluidez da membrana e a sua biodisponibilidade depende do equilíbrio entre a sua síntese e eliminação por espécies reativas de oxigênio. Nós investigamos se o exercício realizado em diferentes intensidades afetaria biodisponibilidade do NO eritrocitário e se levaria a um quadro de estresse oxidativo. Dez homens (26 ± 4 anos, VO2pico 44,1 ± 4,3 mL.kg-1.min-1) realizaram um teste cardiopulmonar máximo em esteira e um teste de exercício submáximo a 70% VO2pico durante 30 min. O sangue foi coletado em repouso e imediatamente após os exercícios para isolamento dos eritrócitos. O exercício máximo aumentou a contagem de eritrócitos, hemoglobina e hematócrito, sem levar a qualquer alteração na massa corporal que pudesse sugerir hemoconcentração devido a uma redução do volume plasmático. Observou-se uma diminuição do influxo de L-arginina depois do teste submáximo, mas não no teste máximo. No entanto, a atividade da óxido nítrico sintase, ou seja, a produção de NO, foi aumentada após o teste máximo. Os níveis de GMPc não se alteraram após ambos os teste de exercício. Em relação aos biomarcadores de estresse oxidativo, o exercício submáximo reduziu a oxidação proteica e aumentou a atividade da catalase e a expressão da glutationa peroxidase, enquanto que o exercício máximo levou a uma maior peroxidação lipídica e diminuição da atividade da SOD. Nem a atividade glutationa peroxidase ou a expressão NADPH oxidase foram afetadas pelo exercício. Estes resultados sugerem que o exercício induziu alterações no estresse oxidativo de eritrócitos, que parecem estar mais associadas com a intensidade do que a duração do execicio. Além disso, nas intensidades recomendadas para a promoção da saúde, o exercício mostrou ser protetor, aumentando a atividade e a expressão de enzimas antioxidantes importantes e reduzindo os danos oxidativos.

Arginine availability and activation of arginine-related pathways at cancer sites have profound effects on the tumor microenvironment, far beyond their well-known role in the hepatic urea cycle. Arginine metabolism impacts not only malignant cells but also the surrounding immune cells behavior, modulating growth, survival, and immunosurveillance mechanisms, either through an arginase-mediated effect on polyamines and proline synthesis, or by the arginine/nitric oxide pathway in tumor cells, antitumor T-cells, myeloid-derived suppressor cells, and macrophages. This review presents evidence concerning the impact of arginine metabolism and arginase activity in the prostate cancer microenvironment, highlighting the recent advances in immunotherapy, which might be relevant for prostate cancer. Even though further research is required, arginine deprivation may represent a novel antimetabolite strategy for the treatment of arginine-dependent prostate cancer.

p53 is a tumour suppressor gene that has been explored for cancer gene therapy as a possible alter- native to the common treatments. The use of plasmid DNA (pDNA) to carry the therapeutic gene has been considered, but it is requisite to preserve its supercoiled (sc) structure, for eliciting a more effective gene expression and therapeutic action. The purification of the sc pDNA using amino acids-based affinity chromatography has been successfully applied, exploring different amino acids and supports. From these studies, it stood out the selectivity of arginine for the recognition of sc pDNA. However, some limitation on the binding capacity was found in the arginine-agarose support, and in the case of monoliths, some fouling and clogging can limit sequential runs. By using macroporous support modified with arginine it was expected to take advantage of the selectivity of the ligand combined with the flow properties and binding capacity offered by the support. The arginine-modified macroporous support was characterized by SEM, EDX and FTIR also to verify the correct immobilization of arginine, and then used for pDNA pu- rification. The support showed to be effective on the sc p53-pDNA isolation, and the robustness was also achieved by accomplishing the purification of plasmids with different sizes, only by slightly adjusting the experimental conditions. Regarding the dynamic binding capacity of the arginine-modified macrop- orous support, it was achieved an improvement of more than 50% in the pDNA binding capacity when compared with their homologous arginine-agarose commercial matrix, suggesting potential economic fea- sibility in case of scale-up.