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Inhibition of systemic inflammation has been a beneficial strategy in treating several non-communicable diseases, which represent one of the major causes of mortality in the world. The Peroxisome Proliferator-Activated Receptors (PPAR) are interesting pharmacological targets, since they can act both through the metabolic and anti-inflammatory pathways. Morus nigra L. has flavonoids in its chemical composition with recognized anti-oxidant activity and often associated with anti-inflammatory activity. Therefore, this study aimed to evaluate the hydroethanolic extract of M. nigra leaves' ability to activate PPAR and promote anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated murine macrophage cells. The leaf extract was prepared by cold maceration, and the chemical profile was obtained by HPLC-DAD. Activation of PPAR α and γ was evaluated by the luciferase reporter assay. The anti-inflammatory activity was assessed by measuring the reactive oxygen species (ROS), nitric oxide (NO), and Tumor Necrosis Factor-α (TNF-α) in RAW 264.7 cells after stimulation with LPS from Escherichia coli. The HPLC-DAD analysis identified two major compounds: rutin and isoquercitrin. The extract showed agonist activity for the two types of PPAR, α and γ, although its major compounds, rutin and isoquercitrin, did not significantly activate the receptors. In addition, the extract significantly reduced the production of ROS, NO, and TNF-α. Treatment with the specific PPAR-α antagonist, GW 6471, was able to partially block the anti-inflammatory effect caused by the extract.

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The enzyme L-asparaginase (L-ASNase) is used in the treatment of Acute Lymphoblastic Leukemia. The preparations of this enzyme for clinical use are derived from bacterial sources and its use is associated with serious adverse reactions. In this context, it is important to find new sources of L-ASNase. In this work, the Placket-Burman Experimental Design (PBD) was used to determine the influence of the variables on the L-ASNase production then it was followed by a 28-4 Factorial Fractional Design (FFD). The results obtained from PBD have shown a range of L-ASNase activity, from 0.47 to 1.77 U/gcell and the results obtained from FFD have showed a range of L-ASNase activity, from 1.10 to 2.36 U/gcell. L-proline and ammonium sulfate were identified as of significant positive variables on this production enzyme by Penicillium cerradense sp. nov. The precise identification of this new species was confirmed by morphological characteristics and sequence comparisons of the nuclear 18S-5.8S-28S partial nrDNA including the ITS1 and ITS2 regions, RNA polymerase II, ß-tubulin and calmodulin genomic regions. The genetic sequence coding for the L-ASNase was obtained after carrying out a full genome sequencing. The L-ASNase expressed by P. cerradense sp. nov may have promising antineoplastic properties.

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The objective of this study was to evaluate biological and phytochemical properties of the aqueous extract from the leaves of Miconia chamissois Naudin (AEMC). Phytochemical properties were assessed by analyzing the chromatographic profile and the polyphenol content of AEMC. Biological properties evaluation was conducted based on cytotoxicity assay and by evaluating the antioxidant, antimicrobial, and enzymatic inhibition activities. Results indicated the presence of phytochemicals in AEMC such as flavonoids and polyphenols, including rutin, isoquercitrin and vitexin derivatives. AEMC showed antioxidant activity, which may be attributed to the high polyphenolic content. Moreover, AEMC demonstrated in vitro enzyme inhibition activity against tyrosinase and alpha-amylase, as well as showed low cytotoxicity. On the other hand, AEMC exhibited weak antimicrobial activity against S. aureusand C. albicans. Thus, AEMC is a promising alternative in search of potential drugs for the treatment of diseases induced by oxidative stress and inflammation, conditions due to hyperpigmentation processes, such as melisma, as well as for diabetes.

El objetivo de este estudio fue detectar las propiedades biológicas y fitoquímicos del extracto acuoso de las hojas de Miconia chamissois Naudin (AEMC). Las propiedades fitoquímicas se evaluaron analizando el perfil cromatográfico y el contenido de polifenoles de AEMC. La evaluación de las propiedades biológicas se realizó en base al ensayo de citotoxicidad y evaluando las actividades de inhibición antioxidante, antimicrobiana y enzimática. Los resultados indicaron la presencia de fitoquímicos en AEMC, como flavonoides y polifenoles, que incluyen derivados de rutina, isoquercitrina y vitexina. AEMC mostró una actividad antioxidante considerable, que puede atribuirse al alto contenido polifenólico. Además, AEMC exhibió actividad de inhibición enzimática in vitro contra tirosinasa y alfa-amilasa, así como mostró baja citotoxicidad. Por otro lado, AEMC demostró actividad antimicrobiana débil contra S. aureusy C. albicans. Por lo tanto, AEMC es una alternativa prometedora en busca de posibles drogas para el tratamiento de enfermedades inducidas por el estrés oxidativo y la inflamación, afecciones debidas a procesos de hiperpigmentación, como el melasma, así como para la diabetes.

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Filamentous fungus Purpureocillium lilacinum is an emerging pathogen that infects immunocompromised and immunocompetent individuals and is resistant to several azole molecules. Although azole resistance mechanisms are well studied in Aspergillus sp. and Candida sp., there are no studies to date reporting P. lilacinum molecular response to these molecules. The aim of this study was to describe P. lilacinum molecular mechanisms involved in antifungal response against fluconazole and itraconazole. Transcriptomic analyses showed that gene expression modulation takes place when P. lilacinum is challenged for 12 h with fluconazole (64 µg/mL) or itraconazole (16 µg/mL). The antifungals acted on the ergosterol biosynthesis pathway, and two homologous genes coding for cytochrome P450 51 enzymes were upregulated. Genes coding for efflux pumps, such as the major facilitator superfamily transporter, also displayed increased expression in the treated samples. We propose that P. lilacinum develops antifungal responses by raising the expression levels of cytochrome P450 enzymes and efflux pumps. Such modulation could confer P. lilacinum high levels of target enzymes and could lead to the constant withdrawal of antifungals, which would force an increase in the administration of antifungal medications to achieve fungal morbidity or mortality. The findings in this work could aid in the decision-making for treatment strategies in cases of P. lilacinum infection.

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OBJECTIVE: To develop recombinant factor IX (FIX) variants with augmented clotting activity. RESULTS: We generated three new variants, FIX-YKALW, FIX-ALL and FIX-LLW, expressed in SK-Hep-1 cells and characterized in vitro and in vivo. FIX-YKALW showed the highest antigen expression level among the variants (2.17 µg-mL), followed by FIX-LLW (1.5 µg-mL) and FIX-ALL (0.9 µg-mL). The expression level of FIX variants was two-five fold lower than FIX-wild-type (FIX-WT) (4.37 µg-mL). However, the biological activities of FIX variants were 15-31 times greater than FIX-WT in the chromogenic assay. Moreover, the new variants FIX-YKALW, FIX-LLW and FIX-ALL also presented higher specific activity than FIX-WT (17, 20 and 29-fold higher, respectively). FIX variants demonstrated a better clotting time than FIX-WT. In hemophilia B mice, we observed that FIX-YKALW promoted hemostatic protection. CONCLUSION: We have developed three improved FIX proteins with potential for use in protein replacement therapy for hemophilia B.

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L-asparaginase has been used in the remission of malignant neoplasms such as acute lymphoblastic leukemia. The search for new sources of this enzyme has become attractive for therapeutics. Traditional methods for biomolecule purification involve several steps. A two-phase system may be a good strategy to anticipate one of these stages. This study aimed to produce and purify a fungal L-asparaginase through an aqueous two-phase micellar system (ATPMS) using Triton X-114. The fungus Penicillium sp.-encoded 2DSST1 was isolated from Cerrado soil. Plackett-Burman design followed by a 24 full factorial design was used to determine the best conditions to produce L-asparaginase. The evaluated variables were L-asparagine, L-proline, wheat bran, potato dextrose broth, ammonium sulfate, yeast extract, sucrose and glucose concentrations, incubation temperature, incubation period, and initial pH of the culture medium. L-asparaginase quantification was valued by the formation of ß-aspartyl hydroxamate. The significant positive variables, L-asparagine, L-proline, potato dextrose broth, and sucrose concentrations, were evaluated at 2 levels (+ 1 and - 1) with triplicate of the central point. After 34 runs, maximum activity (2.33 IU/mL) was achieved at the factorial design central point. A central composite design was performed in ATPMS at two levels (+ 1 and - 1) varying Triton X-114 concentration (w/v), separation phase temperature, and crude extract concentration (w/v). The L-asparaginase partition coefficient (K) was considered the experimental design response. Out of the 16 systems that were examined, the most promising presented a purification factor of 1.4 and a yield of 100%.

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ß-Galactosidases are widely used for industrial applications. These enzymes could be used in reactions of lactose hydrolysis and transgalactosylation. The objective of this study was the production, purification, and characterization of an extracellular ß-galactosidase from a filamentous fungus, Aspergillus niger. The enzyme production was optimized by a factorial design. Maximal ß-galactosidase activity (24.64 U/mL) was found in the system containing 2% of a soybean residue (w/v) at initial pH 7.0, 28 °C, 120 rpm in 7 days. ANOVA of the optimization study indicated that the response data on temperature and pH were significant (p < 0.05). The regression equation indicated that the R2 is 0.973. Ultrafiltration at a 100 and 30 kDa cutoff followed by gel filtration and anion exchange chromatography were carried out to purify the fungal ß-galactosidase. SDS-PAGE revealed a protein with molecular weight of approximately 76 kDa. The partially purified enzyme showed an optimum temperature of 50 °C and optimum pH of 5.0, being stable under these conditions for 15 h. The enzyme was exposed to conditions approaching gastric pH and in pepsin's presence, 80% of activity was preserved after 2 h. These results reveal a A. niger ß-galactosidase obtained from residue with favorable characteristics for food industries.

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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.

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Enzymatic hydrolysis is an important but expensive step in the process to obtain enzyme derived products. Thus, the production of efficient enzymes is of great interest for this biotechnological application. The production of xylanase by Aspergillus foetidus in soybean residues was optimized using 2 × 23 factorial designs. The experimental data was fitted into a polynomial model for xylanase activity. Statistical analyses of the results showed that variables pH and the interaction of pH and temperature had influenced the production of xylanase, with the best xylanase production level (13.98 U/mL) occurring at fermentation for 168 hours, pH 7.0, 28°C, and 120 rpm.

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INTRODUCTION: In 2015, it was observed a rise in the number of microcephalic newborns associated with a history of non-specific febrile sickness and rash during pregnancy in Brazil. Since then, microcephaly has emerged as a public health concern. A few months after, the causal relation between congenital microcephaly and the Zika virus was discovered. Zika virus, an arbovirus, is a new TORCH member that leads to congenital infection through vertical transmission and harms the developing brain, disrupting synaptogenesis, and causing other central nervous system lesions. OBJECTIVE: The purpose of this article is to report the congenital Zika syndrome (CZS) and to emphasize the need for follow-up of the affected children to better know the evolutionary history of this new agent and to optimize the provision of healthcare and improve the quality of life of these patients. METHODS: We review the most relevant literature about clinical manifestations and neuroimaging findings related to neurotropism of Zika virus to characterize the congenital Zika syndrome and suggest the systematization of some exams and procedures to evaluate children exposed to ZIKV with or without microcephaly, according to the author's own experience. CONCLUSIONS: Vertical ZIKV infection can cause a wide spectrum of neurological manifestations that go beyond microcephaly, and even the non-microcephalic child should be followed during the first years of life, because infection may be asymptomatic or lead to neuropsicomotor delay, epilepsy, and visual abnormalities. The appropriate prospective multidisciplinary follow-up of these patients aims to understand the natural history of this new agent and to provide a better development and quality of life for them and their families.

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The gamma-carboxylated proteins belong to a family of proteins that depend on vitamin K for normal biosynthesis. The major representative gamma-carboxylated proteins are the coagulation system proteins, for example, factor VII, factor IX, factor X, prothrombin, and proteins C, S, and Z. These molecules have harbored posttranslational modifications, such as glycosylation and gamma-carboxylation, and for this reason they need to be produced in mammalian cell lines. Human cells lines have emerged as the most promising alternative to the production of gamma-carboxylated proteins. In this chapter, the methods to generate human cells as a platform to produce gamma-carboxylated proteins, for example the coagulation factors VII and IX, are presented. From the cell line modification up to the vitamin K adaptation of the produced cells is described in the protocols presented in this chapter.

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Melanogenesis is a process responsible for melanin production, which is stored in melanocytes containing tyrosinase. Inhibition of this enzyme is a target in the cosmetics industry, since it controls undesirable skin conditions such as hyperpigmentation due to the overproduction of melanin. Species of the Morus genus are known for the beneficial uses offered in different parts of its plants, including tyrosinase inhibition. Thus, this project aimed to study the inhibitory activity of tyrosinase by extracts from Morus nigra leaves as well as the characterization of its chromatographic profile and cytotoxicity in order to become a new therapeutic option from a natural source. M. nigra leaves were collected, pulverized, equally divided into five batches and the standardized extract was obtained by passive maceration. There was no significant difference between batches for total solids content, yield and moisture content, which shows good reproducibility of the extraction process. Tyrosinase enzymatic activity was determined for each batch, providing the percentage of enzyme inhibition and IC50 values obtained by constructing dose-response curves and compared to kojic acid, a well-known tyrosinase inhibitor. High inhibition of tyrosinase activity was observed (above 90% at 15.625 µg/mL). The obtained IC50 values ranged from 5.00 µg/mL ± 0.23 to 8.49 µg/mL ± 0.59 and were compared to kojic acid (3.37 µg/mL ± 0.65). High Performance Liquid Chromatography analysis revealed the presence of chlorogenic acid, rutin and, its major compound, isoquercitrin. The chromatographic method employed was validated according to ICH guidelines and the extract was standardized using these polyphenols as markers. Cytotoxicity, assessed by MTT assay, was not observed on murine melanomas, human keratinocytes and mouse fibroblasts in tyrosinase IC50 values. This study demonstrated the potential of M. nigra leaf extract as a promising whitening agent of natural source against skin hyperpigmentation.

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Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

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Ligation-mediated-PCR was performed followed by the mapping of 177 and 150 integration sites from HepG2 and Hek293 transduced with chimera vector carrying recombinant human Factor IX (rhFIX) cDNA, respectively. The sequences were analyzed for chromosome preference, CpG, transcription start site (TSS), repetitive elements, fragile sites and target genes. In HepG2, rhFIX was had an increased preference for chromosomes 6 and 17; the median distance to the nearest CpG islands was 15,240 base pairs and 37 % of the integrations occurred in RefSeq genes. In Hek293, rhFIX had an increased preference for chromosome 5; the median distance to the nearest CpG islands was 209,100 base pairs and 74 % of the integrations occurred in RefSeq genes. The integrations in both cell lines were distant from the TSS. The integration patterns associated with this vector are different in each cell line.

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The most important properties of a protein are determined by its primary structure, its amino acid sequence. However, protein features can be also modified by a large number of posttranslational modifications. These modifications can occur during or after the synthesis process, and glycosylation appears as the most common posttranslational modification. It is estimated that 50% of human proteins have some kind of glycosylation, which has a key role in maintaining the structure, stability, and function of the protein. Besides, glycostructures can also influence the pharmacokinetics and immunogenicity of the protein. Although the glycosylation process is a conserved mechanism that occurs in yeast, plants, and animals, several studies have demonstrated significant differences in the glycosylation pattern in recombinant proteins expressed in mammalian, yeast, and insect cells. Thus, currently, important efforts are being done to improve the systems for the expression of recombinant glycosylated proteins. Among the different mammalian cell lines used for the production of recombinant proteins, a significant difference in the glycosylation pattern that can alter the production and/or activity of the protein exists. In this context, human cell lines have emerged as a new alternative for the production of human therapeutic proteins, since they are able to produce recombinant proteins with posttranslational modifications similar to its natural counterpart and reduce potential immunogenic reactions against nonhuman epitopes. This chapter describes the steps necessary to produce a recombinant glycoprotein in a human cell line in small scale and also in bioreactors.

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OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO) and baby hamster kidney cells (BHK). Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K. METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones. RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes) was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293. CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.

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Patients with leprosy may have only nerve involvement without skin changes. These cases are known as pure neural leprosy and can be seen in 10% of leprosy patients. Most patients have mononeuritic or multiple mononeuritic neuropathy patterns. The isolated lesion of the superficial peroneal nerve is uncommonly seen. We report a patient with involvement of this nerve in which there was no thickening of superficial nerves. The performed nerve biopsy showed inflammatory infiltration, loss of fibers and presence of Mycobacterium leprae. We believe that in prevalent leprosy countries we should take in mind the possibility of isolated pure neural leprosy in some patients without skin lesion. In these cases the diagnosis of leprosy is impossible on clinical grounds and nerve biopsy is mandatory.

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