Monitoring the production of AAV vectors in insect cells by fluorescence spectroscopy

Adeno-associated viruses (AAV) are among the most promising viral vectors for gene therapy, since they can transduce non-dividing cells from several tissues while maintaining a long-term gene expression. Besides, AAVs possess low immunogenicity compared to other viral vectors, and are physically resistant, which makes them resilient to industrial manufacturing conditions, long-term storage, and in vivo administration. One of the systems available for large scale production of AAVs is the insect cell-baculovirus expression vector system (IC-BEVS). Insect cells grow in suspension to high cell densities with modest growth requirements and without the need of serum supplementation. Consequently, scaling up the production in order to achieve the large number of AAV needed for clinical trials is more straight‑forward than with transfection-based systems. However, methods for online monitoring of AAV production are still lacking. Such methods would allow determination of the best time of harvest in real-time, thus allowing recovery of AAV as soon as its concentration medium was higher. Here we apply Fluorescence Spectroscopy to baculovirus-infected insect cell cultures producing adeno‑associated virus vectors, correlating the spectra to critical process parameters like cell concentration, viability and AAV concentration. Sf9 cells were co-infected with two baculovirus (expressing AAV rep and cap and a CMV-GFP transgene) at low or high multiplicities of infection (MOI), and the culture was followed by Fluorescence Spectroscopy in situ through a bioreactor probe. After an exploratory calibration using data from only one bioreactor, we attested the aptitude of this technique to capture overall data trend: using a 3 component PLS model, we have obtained a calibration NRMSE of 2.9% for total AAV particles per cell, 5.9% for viable cell density and 0.9% for viability). Additional bioreactor productions using different infection parameters (CCI, MOI, time of infection) allowed testing the robustness of fluorescence monitoring to process variability. With this dataset, we tested several pre-treatment methods for the raw spectra, as well as different regression algorithms in order to establish a good predictive model. Ultimately, fluorescence spectroscopy provides a simple tool for online monitoring of key process variables in baculovirus-infected insect cell cultures. Acknowledgments: Funding from Fundação para a Ciência e a Tecnologia, projects EXPL/BBBBIO/1129/2013 and Daniel Pais’ PhD research grant PD/BD/105873/2014

Production of β-galactosidase from recombinant Saccharomyces cerevisiae grown on lactose

Improved productivity and costs reduction in fermentation processes may be attained by using flocculating cell cultures. The production of extracellular heterologous β-galactosidase by recombinant flocculating Saccharomyces cerevisiae cells, expressing the lacA gene (coding for β-galactosidase) of Aspergillus niger under the ADHI promotor and terminator in a bioreactor was studied. The effects of lactose concentration and yeast extract concentration on β-galactosidase production in a semi-synthetic medium were analysed. The extracellular β-galactosidase activity increased linearly with increasing initial lactose concentrations (5–150 g dmˉ³ ). β-Galactosidase production also increased with increased yeast extract concentration. During the entire fermentation, no accumulation of the hydrolysed sugars, glucose and galactose, was observed. The catabolic repression of the recombinant strain when cultured in a medium containing equal amounts of glucose and galactose was confirmed. In complete anaerobiosis, the fermentation of lactose resulted in a very slow fermentation pattern with lower levels of β-galactosidase activity. The bioreactor operation together with optimisation of culture conditions (lactose and yeast extract concentration) led to a 21-fold increase in the extracellular β-galactosidase activity produced when compared with preliminary Erlenmeyer fermentations

A Comparative Genomic Analysis of Energy Metabolism in Sulfate Reducing Bacteria and Archaea

The number of sequenced genomes of sulfate reducing organisms (SRO) has increased significantly in the recent years, providing an opportunity for a broader perspective into their energy metabolism. In this work we carried out a comparative survey of energy metabolism genes found in 25 available genomes of SRO. This analysis revealed a higher diversity of possible energy conserving pathways than classically considered to be present in these organisms, and permitted the identification of new proteins not known to be present in this group. The Deltaproteobacteria (and Thermodesulfovibrio yellowstonii) are characterized by a large number of cytochromes c and cytochrome c-associated membrane redox complexes, indicating that periplasmic electron transfer pathways are important in these bacteria. The Archaea and Clostridia groups contain practically no cytochromes c or associated membrane complexes. However, despite the absence of a periplasmic space, a few extracytoplasmic membrane redox proteins were detected in the Gram-positive bacteria. Several ion-translocating complexes were detected in SRO including H+-pyrophosphatases, complex I homologs, Rnf, and Ech/Coo hydrogenases. Furthermore, we found evidence that cytoplasmic electron bifurcating mechanisms, recently described for other anaerobes, are also likely to play an important role in energy metabolism of SRO. A number of cytoplasmic [NiFe] and [FeFe] hydrogenases, formate dehydrogenases, and heterodisulfide reductase-related proteins are likely candidates to be involved in energy coupling through electron bifurcation, from diverse electron donors such as H2, formate, pyruvate, NAD(P)H, β-oxidation, and others. In conclusion, this analysis indicates that energy metabolism of SRO is far more versatile than previously considered, and that both chemiosmotic and flavin-based electron bifurcating mechanisms provide alternative strategies for energy conservation

Isolation and characterization of essential oils from selected plants

The interest in plants as a potential source of bioactive molecules with application in different areas, from cosmetics to pharmaceuticals, to the food industry and perfumes is increasing [1]. Essential oils, also referred to as volatile oils or essences, can be obtained from plants by using different extraction techniques and are isolated and their chemical composition determined by characterization techniques such as NMR spectroscopy, mass spectrometry and different types of chromatography [2]. In general, essential oils are complex mixtures, and may contain from 50 to 200 compounds of different volatilities. They generally have an intense characteristic aroma, are soluble in organic solvents and insoluble or poorly soluble in water. Identification of its composition is only possible in many cases by comparison of chromatographic data and mass spectrometry of authentic samples. The work presented describes the extraction of essential oils from three sources of plant origin (Ginkgo biloba, laurel and rosemary) with various techniques (soxhlet extraction, hydrodistillation and steam distillation). The extracts were isolated, the major compounds identified and characterised by mass spectrometry, 1H- and 13C-NMR, and the spectra obtained were compared/confirmed with the literature.The author acknowledge to the Fundação para a Ciência e Tecnologia (FCT, Portugal) for financial support to the NMR Portuguese network (PTNMR, Bruker Avance III 400-Univ. Minho), and FCT and FEDER (European Fund for Regional Development)-COMPETE-QREN-EU for funds provided through the Chemistry Research Centre of the University of Minho (Ref. UID/QUI/00686/2013 and UID/QUI/0686/2016).info:eu-repo/semantics/publishedVersio

Climatic extremes in Portugal in the 1780s based on documentary and instrumental records

The final stage of the Little Ice Age in Europe was characterized by strong climatic variability. New documentary sources containing information referring to weather and climate are used in this study to reconstruct and to describe climate conditions in Portugal during the 18th century, mainly in the 1780s. Indexation of documentary data concerning hydric and thermal conditions was based on C. Pfister’s methodology and early instrumental data (1780s and 1790s) were used to verify the reconstruction. Precipitation and temperature were highly variable throughout the 18th century: an alternation of extremely hot to extremely cold months was found. Very cold years occurred mostly in the first 2 decades of the 18th century, but several other cold winters were also detected. Precipitation information is far more frequent than for temperature, and allowed yearly and seasonal indexations. The highest variability was detected in the 1730s and the 1780s. The early 1780s were very dry: during the winter and spring of 1781 and the spring of 1782 several drought episodes occurred, as confirmed by ‘pro-pluvia’ rogations. In contrast, heavy precipitation prevailed from 1784 onwards. The year 1786 was the rainiest in Portugal, triggering floods in northwestern and central Portugal. The year of 1788 was extremely wet and rainfall caused floods along the largest rivers: Douro, Mondego and Tagus. A storm that struck north - western Iberia between 23 and 24 February 1788 is analyzed in detailKLIMHIST: Reconstruction and model simulations of past climate in Portugal using documentary and early instrumental sources (17th-19th century) (PTDC/AAC-CLI/119078/2010

Insights on Ultrafiltration-Based Separation for the Purification and Quantification of Methotrexate in Nanocarriers

The evaluation of encapsulation efficiency is a regulatory requirement for the characterization of drug delivery systems. However, the difficulties in efficiently separating nanomedicines from the free drug may compromise the achievement of accurate determinations. Herein, ultrafiltration was exploited as a separative strategy towards the evaluation of methotrexate (MTX) encapsulation efficiency in nanostructured lipid carriers and polymeric nanoparticles. The effect of experimental conditions such as pH and the amount of surfactant present in the ultrafiltration media was addressed aiming at the selection of suitable conditions for the effective purification of nanocarriers. MTX-loaded nanoparticles were then submitted to ultrafiltration and the portions remaining in the upper compartment of the filtering device and in the ultrafiltrate were collected and analyzed by HPLC-UV using a reversed-phase (C18) monolithic column. A short centrifugation time (5 min) was suitable for establishing the amount of encapsulated MTX in nanostructured lipid carriers, based on the assumption that the free MTX concentration was the same in the upper compartment and in the ultrafiltrate. The defined conditions allowed the efficient separation of nanocarriers from the free drug, with recoveries of >85% even when nanoparticles were present in cell culture media and in pig skin surrogate from permeation assays.info:eu-repo/semantics/publishedVersio

Changes in bone Pb accumulation: Cause and effect of altered bone turnover

Notice: this is the author’s version of a work that was accepted for publication in Bone. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Bone, [Vol 64 (2014 Jul)] DOI 10.1016/j.bone.2014.04.021"This paper assesses the magnitude of Pb uptake in cortical and trabecular bones in healthy animals and animals with altered balance in bone turnover, and the impact of exposure to Pb on serum markers of bone formation and resorption. The results reported herein provide physiological evidence that Pb distributes differently in central compartments in Pb metabolism, such as cortical and trabecular bone, in healthy animals and animals with altered balance in bone turnover, and that exposure to Pb does have an impact on bone resorption resulting in OC-dependent osteopenia. These findings show that Pb may play a role in the etiology of osteoporosis and that its concentration in bones varies as a result of altered bone turnover characteristic of this disease, a long standing question in the field. In addition, data collected in this study are consistent with previous observations of increased half-life of Pb in bone at higher exposures. This evidence is relevant for the necessary revision of current physiologically based kinetic models for Pb in humans.

Bacteria bioburden assessment and MRSA colonization of workers and animals from a Portuguese swine production: a case report

Este trabalho foi financiado pelo Concurso Anual para Projetos de Investigação, Desenvolvimento, Inovação e Criação Artística (IDI&CA) 2016 do Instituto Politécnico de Lisboa. Código de referência: IPL/2016/BBIOR-Health_ESTeSLPigs are an important reservoir of livestock-associated bacteria, including methicillin-resistant staphylococcus aureus (MRSA), which constitute a professional hazard for workers in direct contact with these animals with an increased risk of nasal colonization, potentially associated with subsequent clinical diseases and transference of the infection to others. Here we performed a bioburden characterization concerning bacterial prevalence and resistance (MRSA) in workers and animals from a Portuguese swine production as a case study. Air samples were collected through an impaction method. Biological samples were obtained through nasopharyngeal swab procedure. Identification of S. aureus was performed through immunologic tests. We report an exceedingly high prevalence of total bacteria and S.aureus colonization (100%) in workers and animals whereas all of the identified strains were MRSA. Additionally, air samples demonstrated high values of total bacterial concentration. This work raises awareness to the relevance of bioburden monitoring and the requirement to create occupational standards and take effective preventive measures.info:eu-repo/semantics/publishedVersio

Activation pathway to amino acid adducts

Funding: This work was supported in part by Fundação para a Ciência e a Tecnologia (FCT), Portugal (PTDC/QUI-QUI/113910/2009, RECI/QEQ-MED/0330/2012, UID/QUI/00100/2013 and IF/ 01091/2013/CP1163/CT0001), and by Interagency Agreement Y1ES1027 between the National Center for Toxicological Research/Food and Drug Administration and the National Institute of Environmental Health Sciences/National Toxicology Program. The opinions expressed in this paper do not necessarily represent those of the U.S. Food and Drug Administration. RW, ALG, ILM and SGH thank FCT for postdoctoral and doctoral fellowships (SFRH/BPD/70953/2010, SFRH/BD/72301/2010, SFRH/BD/75426/2010 and SFRH/BD/ 80690/2011, respectively). AMM also acknowledges Programa Operacional Potencial Humano from FCT and the European Social Fund (IF/01091/2013), and the LRI Innovative Science Award. We thank the Portuguese NMR and MS networks (IST nodes) for providing access to the facilities.Nevirapine (NVP) is the non-nucleoside HIV-1 reverse transcriptase inhibitor most commonly used in developing countries, both as a component of combined antiretroviral therapy and to prevent mother-to-child transmission of the virus; however, severe hepatotoxicity and serious adverse cutaneous effects raise concerns about its safety. NVP metabolism yields several phenolic derivatives conceivably capable of undergoing further metabolic oxidation to electrophilic quinoid derivatives prone to react with bionucleophiles and initiate toxic responses. We investigated the ability of two phenolic NVP metabolites, 2-hydroxy-NVP and 3-hydroxy-NVP, to undergo oxidation and subsequent reaction with bionucleophiles. Both metabolites yielded the same ring-contraction product upon oxidation with Frémy's salt in aqueous medium. This is consistent with the formation of a 2,3-NVP-quinone intermediate, which upon stabilization by reduction was fully characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. Additionally, we established that the oxidative activation of 2-hydroxy-NVP involved the transient formation of both the quinone and a quinone-imine, whereas 3-hydroxy-NVP was selectively converted into 2,3-NVP-quinone. The oxidations of 2-hydroxy-NVP and 3-hydroxy-NVP in the presence of the model amino acids ethyl valinate (to mimic the highly reactive N-terminal valine of hemoglobin) and N-acetylcysteine were also investigated. Ethyl valinate reacted with both 2,3-NVP-quinone and NVP-quinone-imine, yielding covalent adducts. By contrast, neither 2,3-NVP-quinone nor NVP-derived quinone-imine reacted with N-acetylcysteine. The product profile observed upon Frémy's salt oxidation of 2-hydroxy-NVP in the presence of ethyl valinate was replicated with myeloperoxidase-mediated oxidation. Additionally, tyrosinase-mediated oxidations selectively yielded 2,3-NVP-quinone-derived products, while quinone-imine-derived products were obtained upon lactoperoxidase catalysis. These observations suggest that the metabolic conversion of phenolic NVP metabolites into quinoid electrophiles is biologically plausible. Moreover, the lack of reaction with sulfhydryl groups might hamper the in vivo detoxification of NVP-derived quinone and quinone-imine metabolites via glutathione conjugation. As a result, these metabolites could be available for reaction with nitrogen-based bionucleophiles (e.g., lysine residues of proteins) ultimately eliciting toxic events.publishersversionpublishe

Desenvolvimento, produção e caracterização de nanocristais de fármacos pouco solúveis

Poorly soluble drugs have low bioavailability, representing a major challenge for the pharmaceutical industry. Processing drugs into the nanosized range changes their physical properties, and these are being used in pharmaceutics to develop innovative formulations known as Nanocrystals. Use of nanocrystals to overcome the problem of low bioavailability, and their production using different techniques such as microfluidization or high pressure homogenization, was reviewed in this paper. Examples of drugs, cosmetics and nutraceutical ingredients were also discussed. These technologies are well established in the pharmaceutical industry and are approved by the Food and Drug Administration