Tripping over emerging pathogens around the world: A phylogeographical approach for determining the epidemiology of Porcine circovirus-2 (PCV-2), considering global trading

AbstractPorcine circovirus-2 (PCV-2) is an emerging virus associated with a number of different syndromes in pigs known as Porcine Circovirus Associated Diseases (PCVAD). Since its identification and characterization in the early 1990s, PCV-2 has achieved a worldwide distribution, becoming endemic in most pig-producing countries, and is currently considered as the main cause of losses on pig farms. In this study, we analyzed the main routes of the spread of PCV-2 between pig-producing countries using phylogenetic and phylogeographical approaches. A search for PCV-2 genome sequences in GenBank was performed, and the 420 PCV-2 sequences obtained were grouped into haplotypes (group of sequences that showed 100% identity), based on the infinite sites model of genome evolution. A phylogenetic hypothesis was inferred by Bayesian Inference for the classification of viral strains and a haplotype network was constructed by Median Joining to predict the geographical distribution of and genealogical relationships between haplotypes. In order to establish an epidemiological and economic context in these analyses, we considered all information about PCV-2 sequences available in GenBank, including papers published on viral isolation, and live pig trading statistics available on the UN Comtrade database (http://comtrade.un.org/). In these analyses, we identified a strong correlation between the means of PCV-2 dispersal predicted by the haplotype network and the statistics on the international trading of live pigs. This correlation provides a new perspective on the epidemiology of PCV-2, highlighting the importance of the movement of animals around the world in the emergence of new pathogens, and showing the need for effective sanitary barriers when trading live animals

Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill

<p>Abstract</p> <p>Background</p> <p><it>Eucalyptus globulus </it>and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative reverse transcription-polymerase chain reaction (qPCR); however, reliable use of this technique requires reference genes for normalization. There is no single reference gene that can be regarded as universal for all experiments and biological materials. Thus, the identification of reliable reference genes must be done for every species and experimental approach. The present study aimed at identifying suitable control genes for normalization of gene expression associated with adventitious rooting in <it>E. globulus </it>microcuttings.</p> <p>Results</p> <p>By the use of two distinct algorithms, <it>geNorm </it>and <it>NormFinder</it>, we have assessed gene expression stability of eleven candidate reference genes in <it>E. globulus</it>: <it>18S, ACT2, EF2, EUC12, H2B, IDH, SAND, TIP41, TUA, UBI </it>and <it>33380</it>. The candidate reference genes were evaluated in microccuttings rooted <it>in vitro</it>, in presence or absence of auxin, along six time-points spanning the process of adventitious rooting. Overall, the stability profiles of these genes determined with each one of the algorithms were very similar. Slight differences were observed in the most stable pair of genes indicated by each program: <it>IDH </it>and <it>SAND </it>for <it>geNorm</it>, and <it>H2B </it>and <it>TUA </it>for <it>NormFinder</it>. Both programs indentified <it>UBI </it>and <it>18S </it>as the most variable genes. To validate these results and select the most suitable reference genes, the expression profile of the <it>ARGONAUTE1 </it>gene was evaluated in relation to the most stable candidate genes indicated by each algorithm.</p> <p>Conclusion</p> <p>Our study showed that expression stability varied between putative reference genes tested in <it>E. globulus</it>. Based on the <it>AGO1 </it>relative expression profile obtained using the genes suggested by the algorithms, <it>H2B </it>and <it>TUA </it>were considered as the most suitable reference genes for expression studies in <it>E. globulus </it>adventitious rooting. <it>UBI </it>and <it>18S </it>were unsuitable for use as controls in qPCR related to this process. These findings will enable more accurate and reliable normalization of qPCR results for gene expression studies in this economically important woody plant, particularly related to rooting and clonal propagation.</p

Sustainable lysis of Bacillus subtilis biomass to recover the biopharmaceutical L-asparaginase

The first-line biopharmaceutical used to treat Acute lymphoblastic leukemia (ALL), Oncaspar, is based on the enzyme L-asparaginase (ASNase), and has annual sales of ca. USD $100 million. In addition to other sources, genetically modified Bacillus subtilis is regarded as one of the most promising hosts for the ASNase production. The Aliivibrio fischeri ASNase type II, which has anti-tumour activity due its higher specific affinity for L-asparagine, expressed in B. subtillis is located in the periplasm. Therefore, cell lysis is required for the ASNase recovery. Nevertheless, typical cell lysis approaches, e.g. chemical methods with surfactants lead to some biocompatibility concerns and the need of extra purification steps. To overcome this drawback, in this work, ultrasound sonication (USS) conditions were studied to develop a greener and more biocompatible method for ASNase recovery from B. subtilis cell lysis. The USS cell lysis was optimized regarding the amplitude of USS pulse, number of lysis cycles and mass of cells/volume of solvent ratio. The identification and quantification of ASNase and major impurities present in the cell extract after lysis were investigated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion high-performance liquid chromatography (SE-HPLC). ASNase activity was determined by monitoring the hydrolysis of the substrate, L-asparagine. The results obtained show that the ideal conditions for B. subtilis cell lysis are an amplitude of USS pulse of 60%, 40 cycles of lysis and 10 mL of phosphatebuffered saline (PBS) per 1 g of cells. Overall, an optimized sustainable B. subtilis cell lysis method was developed, avoiding the use of surfactants and with low energy consumption.publishe

Overview on protein extraction and purification using ionic-liquid-based processes

Proteins are one the most widely studied biomolecules with diverse functions and applications. Aiming at overcoming the current drawbacks of purification processes of proteins, the introduction of ionic liquids (ILs) has been a hot topic of research. ILs have been applied in the creation of aqueous biphasic systems (IL-based ABS), solid-phase extractions through poly(ionic liquid)s (PILs) and supported ionic-liquid phases (SILPs), and in the crystallization of proteins. In this sense, ILs have emerged as solvents, electrolytes or adjuvants, or as supported materials to tune the adsorption/affinity capacity aiming at developing an efficient, cost-effective, sustainable and green IL-based process for protein extraction. This review discusses different IL-based processes in the extraction and purification of proteins in the past years, namely IL-based aqueous biphasic systems (IL-based ABS), solid-phase extractions through PILs and SILPs, and protein crystallization. The type and structure of ILs applied and their influence in the different processes performance are also discussed.publishe

Reusability of L-asparaginase immobilized on silica-based supported ionic liquids

L-asparaginase (ASNase) is an aminohydrolase enzyme used as an anticancer drug, e.g. in the treatment of acute lymphoblastic leukemia, in acrylamide reduction and in biosensing. Nevertheless, its low stability and thermolability, and susceptibility to proteases, hinder its application in the health and food industries. Hence, the improvement of its properties through efficient immobilization methods is in high demand. Thus, this work aims the development of silica-based supported ionic liquids (SILs) for the ASNase immobilization to improve its stability and enable its reusability. While activated silica with no ILs only kept total initial ASNase activity during the first cycle of reaction, SILs allowed 5 cycles of reaction, keeping 82% of initial ASNase activity, reinforcing their potential as alternative enzymatic supports.publishe

A flow-through strategy using supported ionic liquids for L-asparaginase purification

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Purification of antileukemic drugs through silica-based supported ionic liquids

L-asparaginase (LA) is an enzyme used as a biopharmaceutical for the treatment of acute lymphoblastic leukemia. LA can be produced via fermentation and its purification usually comprises several steps including precipitation, liquid-liquid extraction and chromatography techniques. Among these, ion exchange chromatography, which is often preceded by precipitation with salts as a first pre-chromatographic step, is the most used. However, theses common strategies for protein purification result in low yields and purity, requiring long processing times, while leading to a consequent increase of the process costs. Therefore, the demand for new cost-effective production/purification processes play now a priority role. This work aims the development of cost-effective technologies to purify LA from the complex fermentation medium from Bacillus Subtillis. Silica-based supported ionic liquids (SILs) are investigated as cost-effective purification materials for the target enzyme. The concentration of the extract from the fermentation, material/ extract from fermentation ratio and contact time effects in the purity and yield of LA were optimized. With this strategy, process costs, energy consumed, and waste generated, may be significantly decreased, which may lead to this biopharmaceutical price decrease and wider application.publishe

Purification of antileukemic drugs through silica-based supported ionic liquids

L-asparaginase (LA) is an enzyme used as a biopharmaceutical for the treatment of acute lymphoblastic leukemia. LA can be produced via fermentation and its purification usually comprises ion exchange chromatography, which is often preceded by precipitation with salts as a first pre-chromatographic step. However, this purification strategy result in low yields and purity, requires long processing times, while leading to a consequent increase of the process costs. Therefore, the demand for new cost-effective purification processes play now a priority role. In this work silica-based supported ionic liquids (SILs) are investigated as an alternative technology to purify LA from the complex fermentation medium from Bacillus subtillis. The concentration of the extract from the fermentation, material/ extract from fermentation ratio and contact time effects in the purity and yield of LA were optimized. With this strategy, process costs, energy consumed, and waste generated, may be significantly decreased, which may lead to this biopharmaceutical price decrease and wider application.publishe

Enhanced enzyme reuse through the bioconjugation of L-asparaginase and silica-based supported ionic liquid-like phase materials

L-asparaginase (ASNase) is an amidohydrolase that can be used as a biopharmaceutical, as an agent for acrylamide reduction, and as an active molecule for L-asparagine detection. However, its free form displays some limitations, such as the enzyme’s single use and low stability. Hence, immobilization is one of the most effective tools for enzyme recovery and reuse. Silica is a promising material due to its low-cost, biological compatibility, and tunable physicochemical characteristics if properly functionalized. Ionic liquids (ILs) are designer compounds that allow the tailoring of their physicochemical properties for a given task. If properly designed, bioconjugates combine the features of the selected ILs with those of the support used, enabling the simple recovery and reuse of the enzyme. In this work, silica-based supported ionic liquid-like phase (SSILLP) materials with quaternary ammoniums and chloride as the counterion were studied as novel supports for ASNase immobilization since it has been reported that ammonium ILs have beneficial effects on enzyme stability. SSILLP materials were characterized by elemental analysis and zeta potential. The immobilization process was studied and the pH effect, enzyme/support ratio, and contact time were optimized regarding the ASNase enzymatic activity. ASNase–SSILLP bioconjugates were characterized by ATR-FTIR. The bioconjugates displayed promising potential since [Si][N3444]Cl, [Si][N3666]Cl, and [Si][N3888]Cl recovered more than 92% of the initial ASNase activity under the optimized immobilization conditions (pH 8, 6 × 10−3 mg of ASNase per mg of SSILLP material, and 60 min). The ASNase–SSILLP bioconjugates showed more enhanced enzyme reuse than reported for other materials and immobilization methods, allowing five cycles of reaction while keeping more than 75% of the initial immobilized ASNase activity. According to molecular docking studies, the main interactions established between ASNase and SSILLP materials correspond to hydrophobic interactions. Overall, it is here demonstrated that SSILLP materials are efficient supports for ASNase, paving the way for their use in the pharmaceutical and food industries.publishe