Nonenzymatic glycation of hemoglobin is a slow, continuous, and irreversible process which takes place during the whole lifespan of the erythrocyte. When hemolytic diseases are ruled out, the levels of glycated hemoglobins reflect the time-averaged serum glucose concentration for the preceding weeks. Canine hemoglobin also binds physiologically to intraerythrocytic glucose to form a glycated fraction which provides information on the animal's long-term glycemic status. This study describes an overall evaluation of ion-exchange microchromatography and thiobarbituric acid (TBA) colorimetry for the measurement of canine glycated hemoglobins. The intra- and inter-assay coefficients of variation (CVs) found were less than 5% in normal and diabetic canine samples, and both assays proved linear over the analytical range tested, which was wide enough to include the expected clinical values. Under our laboratory's conditions, the reference range for HbA(1) was 5.82 +/- 0.62% and for HbA(1)c was 2.35 -/+ 0.47%. Sample stability was lower using the ion-exchange procedure, with increases in HbA(1) observed after 4 days in whole blood and hemolysates stored at room temperature, after 12 days in whole blood stored at 4 degrees C, and after 7 days in hemolysates stored at 4 degrees C and -20 degrees C. In the case of TBA colorimetry, whole blood was stable for at least 21 days at room temperature and at 4 degrees C, and hemolysates were stable for 18 days at room temperature, at least 21 days at 4 degrees C, and up to 3 months at -20 degrees C.

No abstract

No abstract

Rabies is an ancient fatal disease with no other available treatment than post-exposure vaccination, where the bite of infected animals, mainly dogs, is the leading cause of its transmission to human beings. In this context, global vaccination campaigns of companion animals, as well as wildlife reservoirs vaccination, are key factors to achieve the "Zero by 30" plan that pursues the eradication of dog-mediated human rabies by 2030. Rabies virus-neutralizing antibodies (VNAs) play an essential role in the disease protection, as it correlates with an adequate immune response and allows evaluating pre-or post-exposure prophylaxis efficacy. Hence, counting with reliable, accurate, and robust serological tests is of paramount importance. Currently, RFFIT and FAVN are the gold standard VNAs tests recommended by both the WHO and the OIE. Despite these methodologies are efficient and widely used, they present several drawbacks, as they are less easily to standardize and require the use of live rabies virus, containment facilities, and skilled professionals. Thus, in this review, we describe the state-of-the-art of alternative analytical methodologies currently available for rabies serology, with novel approaches based on pseudotyped recombinant viruses and emphasizing in the antigen binding methodologies that detect and quantify antibodies against the rabies glycoprotein. We discussed the wide range of assays that are interesting tools for a faster measurement of anti-rabies glycoprotein antibodies and, in some cases, less complex and more versatile than the gold standard methods. Finally, we discussed the key issues during the design and optimization steps of ELISA assays, highlighting the importance of validation and standardization procedures to improve rabies serology tests and, as a consequence, their results. Key points• An exhaustive revision of rabies serology testing was made.• No rabies serology assay can be thought as better than others for all intents and purposes.• The validation procedure guarantees reliable and consistent results among the globe.

The prokaryotic beta serine recombinase (beta-rec) catalyzes site-specific recombination between two directly oriented six sites (93 bp) in mammalian cells, both in episomal and in chromosomally integrated substrates. The beta-rec/six exclusive intramolecular site-specific recombination (SSR) system has been proposed as a suitable approach when several independently controlled recombination events are needed in a single cell. Here we explored the use of the beta-rec/six system for selective induction of genome-targeted modifications. We generated and analyzed mouse transgenic lines (Tgbeta) expressing beta-rec under the control of the Lck promoter. beta-rec activity was demonstrated, and there was no evidence of alterations to thymic or peripheral T cell development. We developed two transgenic mouse lines harboring different target sequences (Tgrec and KOsix) and analyzed the effect of beta-rec expression on these animals. The results indicate that the beta-rec/six SSR system is functional for in vivo gene-targeting applications.

No abstract

Fabry disease is an X-linked recessive disorder caused by a deficiency in lysosomal α-Galactosidase A. Currently, two enzyme replacement therapies (ERT) are available. However, access to orphan drugs continues to be limited by their high price. Selection of adequate high-expression systems still constitutes a challenge for alleviating the cost of treatments. Several strategies have been implemented, with varying success, trying to optimize the production process of recombinant human α-Galactosidase A (rhαGAL) in Chinese hamster ovary (CHO-K1) cells. Herein, we describe for the first time the application of a strategy based on third-generation lentiviral particles (LP) transduction of suspension CHO-K1 cells to obtain high-producing rhαGAL clones (3.5 to 59.4 pg cell d ). After two purification steps, the active enzyme was recovered (2.4 × 10 U mg ) with 98% purity and 60% overall yield. Michaelis-Menten analysis demonstrated that rhαGAL was capable of hydrolyzing the synthetic substrate 4MU-α-Gal at a comparable rate to Fabrazyme®, the current CHO-derived ERT available for Fabry disease. In addition, rhαGAL presented the same mannose-6-phosphate (M6P) content, about 40% higher acid sialic amount and 33% reduced content of the immunogenic type of sialic acid (Neu5Gc) than the corresponding ones for Fabrazyme®. In comparison with other rhαGAL production processes reported to date, our approach achieves the highest rhαGAL productivity preserving adequate activity and glycosylation pattern. Even more, considering the improved glycosylation characteristics of rhαGAL, which might provide advantages regarding pharmacokinetics, our enzyme could be postulated as a promising alternative for therapeutic use in Fabry disease. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1334-1345, 2017.

In the pharmaceutical industry, the need for high levels of protein expression in mammalian cells has prompted the search for new strategies, including technologies to obtain cells with improved mechanisms that enhance its transcriptional activity, folding, or protein secretion. Chinese Hamster Ovary (CHO) cells are by far the most used host cell for therapeutic protein expression. However, these cells produce specific glycans that are not present in human cells and therefore potentially immunogenic. As a result, there is an increased interest in the use of human‐derived cells for therapeutic protein production. For many decades, human embryonic kidney (HEK) cells were exclusively used for research. However, two products for therapeutic indication were recently approved in the United States. It was previously shown that tethered Magoh, an Exon‐junction complex core component, to specific mRNA sequences, have had significant positive effects on mRNA translational efficiency. In this study, a HEK Magoh‐overexpressing cell line and clones, designated here as HEK‐MAGO, were developed for the first time. These cells exhibited improved characteristics in protein expression, reaching —two‐ to threefold increases in rhEPO protein production in comparison with the wild‐type cells. Moreover, this effect was promoter independent highlighting the versatility of this expression platform.