Diversity and genetic structure of natural populations of the palm tree Euterpe precatoria (Arecaceae) / Diversidad y estructura genética de poblaciones naturales de la palmera Euterpe precatoria (Arecaceae)

Abstract Introduction: The natural ecosystems of northern Mato Grosso, Brazil, are in process of fragmentation, mainly due to population growth and the expansion of agriculture. This endangers the palm Euterpe precatoria (locally known as açaí), used for construction, palm hearts, juices and ice cream. Objective: To evaluate the local diversity and genetic structure in native populations of E. precatoria. Methods: We collected leaves from 106 fruiting palms from five populations in Mato Grosso State, for analysis of microsatellite markers with Polymerase Chain Reaction. Results: The five SSR loci revealed a total of 30 alleles, ranging from 5 (EE23 and EE43) to 7 (EE2 and EE15), with an average of 6 alleles per locus. The mean PIC was 0.74 and confirmed low heterozygosity and inbreeding. The UPGMA dendrogram produced two groups and molecular variance revealed greater genetic differentiation within populations. The high levels of homozygous microsatellite loci indicate low genetic diversity. Conclusions: These populations have low gene diversity, high average number of alleles per locus, and rare and exclusive alleles. We recommend the establishment of permanent conservation units with corridors among them.

Resumen Introducción: Los ecosistemas naturales del norte de Mato Grosso, Brasil, están en proceso de fragmentación, principalmente debido al crecimiento de la población y la expansión de la agricultura. Esto pone en peligro la palma Euterpe precatoria (localmente conocida como açaí), utilizada para la construcción, extracción de palmito, preparación de jugos y helados. Objetivo: Evaluar la diversidad local y estructura genética en poblaciones nativas de E. precatoria. Métodos: Recolectamos hojas de 106 palmas fructíferas de cinco poblaciones en el estado de Mato Grosso, para análisis de marcadores microsatélites con el método de Reacción en Cadena de la Polimerasa (PCR). Resultados: Los cinco loci SSR revelaron un total de 30 alelos, que van desde 5 (EE23 y EE43) hasta 7 (EE2 y EE15), con un promedio de 6 alelos por locus. El PIC medio fue de 0.74 y confirmó baja heterocigosidad y endogamia en las poblaciones. El dendrograma UPGMA produjo dos grupos y la varianza molecular reveló una mayor diferenciación genética dentro de las poblaciones. Los loci de microsatélites presentaron un alto nivel de homocigotos lo que indica una baja diversidad genética. Conclusiones: Estas poblaciones tienen baja diversidad genética, alto promedio de alelos por locus y alelos raros y únicos. Recomendamos el establecimiento de unidades de conservación permanentes con corredores entre ellas.

Neutralizing Antibodies to Human Cytomegalovirus Recombinant Proteins Reduce Infection in an Ex Vivo Model of Developing Human Placentas.

Human cytomegalovirus (HCMV) is the leading viral cause of congenital disease and permanent birth defects worldwide. Although the development of an effective vaccine is a public health priority, no vaccines are approved. Among the major antigenic targets are glycoproteins in the virion envelope, including gB, which facilitates cellular entry, and the pentameric complex (gH/gL/pUL128-131), required for the infection of specialized cell types. In this study, sera from rabbits immunized with the recombinant pentameric complex were tested for their ability to neutralize infection of epithelial cells, fibroblasts, and primary placental cell types. Sera from rhesus macaques immunized with recombinant gB or gB plus pentameric complex were tested for HCMV neutralizing activity on both cultured cells and cell column cytotrophoblasts in first-trimester chorionic villus explants. Sera from rabbits immunized with the pentameric complex potently blocked infection by pathogenic viral strains in amniotic epithelial cells and cytotrophoblasts but were less effective in fibroblasts and trophoblast progenitor cells. Sera from rhesus macaques immunized with the pentameric complex and gB more strongly reduced infection in fibroblasts, epithelial cells, and chorionic villus explants than sera from immunization with gB alone. These results suggest that the pentameric complex and gB together elicit antibodies that could have potential as prophylactic vaccine antigens.

Estimation of Nuwiq® (simoctocog alfa) activity using one-stage and chromogenic assays-Results from an international comparative field study.

BACKGROUND: Accurate determination of coagulation factor VIII activity (FVIII:C) is essential for effective and safe FVIII replacement therapy. FVIII: C can be measured by one-stage and chromogenic substrate assays (OSAs and CSAs, respectively); however, there is significant interlaboratory and interassay variability. AIMS: This international comparative field study characterized the behaviour of OSAs and CSAs used in routine laboratory practice to measure the activity of Nuwiq® (human-cl rhFVIII, simoctocog alfa), a fourth-generation recombinant human FVIII produced in a human cell line. METHODS: FVIII-deficient plasma was spiked with Nuwiq® or Advate® at 1, 5, 30 and 100 international units (IU)/dL. Participating laboratories analysed the samples using their routine procedures and equipment. Accuracy, inter- and intralaboratory variation, CSA:OSA ratio and the impact of different OSA and CSA reagents were assessed. RESULTS: Forty-nine laboratories from 9 countries provided results. Mean absolute FVIII:C was comparable for both products at all concentrations with both OSA and CSA, with interproduct ratios (Nuwiq® :Advate® ) of 1.02-1.13. Mean recoveries ranged from 97% to 191% for Nuwiq® , and from 93% to 172% for Advate® , with higher recoveries at lower concentrations. Subgroup analyses by OSA and CSA reagents showed minor variations depending on reagents, but no marked differences between the two products. CSA:OSA ratios based on overall means ranged from 0.99 to 1.17 for Nuwiq® and from 1.01 to 1.17 for Advate® . CONCLUSIONS: Both OSAs and CSAs are suitable for the measurement of FVIII:C of Nuwiq® in routine laboratory practice, without the need for a product-specific reference standard.

Genetic divergences between Spondias mombin (Anacardiaceae) genotypes found through morphological traits / Divergencia genética entre genotipos de Spondias mombin (Anacardiaceae) según caracteres morfológicos

Abstract The species Spondias mombin, is native to the Amazonian region. Since these trees' cultivation is incipient, their exploitation is done through extraction techniques. The aim of the present study was to assess the genetic divergences between S. mombin genotypesand to quantify the relative contribution from 12 morphological traits of the species' fruits and seeds, as well as to collect data able to subsidize future research on the species conservation and domestication. 60 genotypes were assessed in total, and ten fruits of each genotype were analyzed. Eight descriptors were used for fruit characterization, namely: fruit mass, pulp weight, volume, length, width, thickness, total soluble solids content and hydrogenionic potential. The seed descriptors were mass, length, width and thickness. The data were assessed through the principal components and groupings by applying the UPGMA and Tocher methods. They were analyzed in the GENES software, based on the dissimilarity matrix (Euclidean distance average). The analysis applied to the principal components showed that the first three components explained 83 % accumulated variation. The main traits contributing to the genotype discrimination were fruit width, fruit pulp weight, pH, seed length and thickness, and the most responsive traits to S. mombin genotype selection. The features fruit mass, seed width, fruit thickness, fruit volume, fruit length, seed mass and total soluble solid content presented the smallest contribution to diversity. The grouping methods UPGMA and Tocher evidenced genetic divergence between the analyzed genotypes. Genotypes 37 and 41 were more divergent than the others, what makes them promising for crossings in future genetic enhancement programs focused on the species' domestication.

Resumen Spondias mombin es un árbol nativo de la región amazónica; su explotación se produce de manera extractiva, siendo aún incipiente el cultivo de la especie. El objetivo de este estudio fue evaluar la divergencia genética entre genotipos de S. mombin y cuantificar la contribución relativa de 12 características morfológicas de frutos y semillas, para obtener datos que permitan investigaciones futuras relativas a la conservación y domesticación de la especie. Se evaluaron 60 genotipos; de cada uno se analizaron diez frutos. Los descriptores utilizados para la caracterización de los frutos fueron ocho: masa del fruto, peso de la pulpa, volumen, longitud, anchura, espesor, contenido total de sólidos solubles y pH; los descriptores de semillas fueron cuatro: masa, longitud, anchura y espesor. Los datos se evaluaron por medio de componentes principales y agrupaciones obtenidas por métodos UPGMA y Tocher, de la matriz de disimilitud (distancia euclideana media) con ayuda del programa GENES. A través del análisis de los componentes principales, se verificó que los tres primeros componentes explican 83.63 % de la variación acumulada. Las características que más contribuyen a la discriminación de los genotipos fueron: anchura del fruto, peso de la pulpa, pH, longitud y espesor de semillas, y son las más significativas para la selección de genotipos de S. mombin. Las contribuciones más pequeñas a la diversidad se deben a: la masa del fruto, anchura de semilla, espesor, volumen y longitud del fruto, así como a la masa de las semillas y el contenido de sólidos solubles totales. Ambos métodos de agrupación, UPGMA y Tocher, revelaron que existe divergencia genética entre los genotipos analizados. Los genotipos 37 y 41 son los más divergentes respecto a los otros; por eso son ideales para programas de mejoramiento genético con miras a la domesticación de la especie.

Rational design and optimization of downstream processes of virus particles for biopharmaceutical applications: current advances.

The advent of advanced therapies in the pharmaceutical industry has moved the spotlight into virus-like particles and viral vectors produced in cell culture holding great promise in a myriad of clinical targets, including cancer prophylaxis and treatment. Even though a couple of cases have reached the clinic, these products have yet to overcome a number of biological and technological challenges before broad utilization. Concerning the manufacturing processes, there is significant research focusing on the optimization of current cell culture systems and, more recently, on developing scalable downstream processes to generate material for pre-clinical and clinical trials. We review the current options for downstream processing of these complex biopharmaceuticals and underline current advances on knowledge-based toolboxes proposed for rational optimization of their processing. Rational tools developed to increase the yet scarce knowledge on the purification processes of complex biologicals are discussed as alternative to empirical, "black-boxed" based strategies classically used for process development. Innovative methodologies based on surface plasmon resonance, dynamic light scattering, scale-down high-throughput screening and mathematical modeling for supporting ion-exchange chromatography show great potential for a more efficient and cost-effective process design, optimization and equipment prototyping.

Large-scale production and purification of VLP-based vaccines.

Virus-like particles (VLPs) hold tremendous potential as vaccine candidates. These innovative biopharmaceuticals present the remarkable advantages of closely mimicking the three-dimensional nature of an actual virus while lacking the virus genome packaged inside its capsid. As a result, an equally efficient but safer prophylaxis is anticipated as compared to inactivated or live attenuated viral vaccines. With the advent of successful cases of approved VLP-based vaccines, pharmaceutical companies are indeed redirecting their resources to the development of such products. This paper reviews the current choices and trends of large-scale production and purification of VLP-based vaccines generated through the baculovirus expression vector system using insect cells.

Quality control and analytical methods for baculovirus-based products.

Recombinant baculoviruses (rBac) are used for many different applications, ranging from bio-insecticides to the production of heterologous proteins, high-throughput screening of gene functions, drug delivery, in vitro assembly studies, design of antiviral drugs, bio-weapons, building blocks for electronics, biosensors and chemistry, and recently as a delivery system in gene therapy. Independent of the application, the quality, quantity and purity of rBac-based products are pre-requisites demanded by regulatory authorities for product licensing. To guarantee maximization utility, it is necessary to delineate optimized production schemes either using trial-and-error experimental setups ("brute force" approach) or rational design of experiments by aid of in silico mathematical models (Systems Biology approach). For that, one must define all of the main steps in the overall process, identify the main bioengineering issues affecting each individual step and implement, if required, accurate analytical methods for product characterization. In this review, current challenges for quality control (QC) technologies for up- and down-stream processing of rBac-based products are addressed. In addition, a collection of QC methods for monitoring/control of the production of rBac derived products are presented as well as innovative technologies for faster process optimization and more detailed product characterization.

Impact of ligand density on the optimization of ion-exchange membrane chromatography for viral vector purification.

The effect of ligand density on anion-exchange membrane chromatography (AEXmc) for the purification of recombinant baculoviruses (rBVs), potential viral vectors in clinical applications, is studied by surface plasmon resonance on customized AEX surfaces and gradient elution experiments on Sartobind D membrane prototypes with different diethylamine ligand densities, complemented by dynamic light scattering analysis for estimation of the hydrodynamic particle size of the various biologics. A chromatographic-column model based on the steric mass action model of ion exchange is employed to analyze the gradient-elution AEXmc experiments, extrapolate the results to other operating conditions, and provide directions for process improvement. Although counterintuitively, the experimental evidence provided in this study shows that the lowering of ligand density is beneficial for rBV purification by AEXmc in bind-and-elute-mode, because it decreases the residual concentrations of host cell protein, dsDNA, and non-infective rBVs in the eluted product cut, and increases the overall yield by roughly 20% over current standard values. Overall, we present a case study on how rational design can streamline downstream process development.

Analysis of adsorption of a baculovirus bioreaction bulk on an ion-exchange surface by surface plasmon resonance.

The binding and elution of the key components of a bioreaction bulk for the production of recombinant baculoviruses-a promising viral vector for gene therapy and vaccination-on a model ion-exchange surface have been successfully measured and interpreted by surface plasmon resonance (SPR) spectroscopy. The micro-scaled, ion-exchange surface was produced by immobilizing a typical ion-exchange ligand, diethylaminoethyl, onto commercially available planar gold sensor chip surfaces, which were pre-derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid. Each isolated analyte was injected into the SPR cell at defined operating conditions of salt and solute concentrations to determine the adsorption equilibrium plateau, and then eluted at the same salt concentration, upon which a well-defined, residual desorption equilibrium plateau was observed. From the analysis of the binding and elution curves and equilibrium plateaus for seven key biomolecules, it is possible to determine the adsorption isotherms over a broad range of equilibrium conditions for the three main cuts of the baculovirus bioreaction bulk: the product (the infective baculovirus), the main product-related impurities, and the main process-related impurities. A model that quantitatively explains the SPR-derived adsorption/desorption data was successfully developed for this complex biological system.

Modeling electrostatic interactions of baculovirus vectors for ion-exchange process development.

Product-related impurities constitute a major burden in the production of recombinant viral vectors for gene therapy and vaccination; it impairs not only the biological efficacy of the preparation but the process yield/productivity. Recombinant baculovirus was used as an enveloped virus model to address this issue. Given that ion-exchange chromatography is a process of choice for purification of viral vectors, the analysis of the electrostatic behavior can be instrumental for the improvement of impurity removal. The main species, product (infective virus particle) and product-derived impurities (dsDNA-, glycoprotein-, and envelope-deprived baculovirus particles), were isolated and correspondent zeta potentials were analyzed through dynamic light scattering. A model of the virus based on the viral components critical for biological function is proposed. The contribution of these viral components to the overall particle electrostatic interaction energy profile (calculated between the particle and a putative ion-exchange surface) was assessed as a function of ionic strength and pH. This resulted in a deterministic tool capable of distinguishing the electrostatic properties of the infective virus particle from the major virus-related impurities. Within an ion-exchange bind-elute process, this knowledge helps narrow the optimization space in early stage process development for viral vectors by predicting the best selectivity conditions.

Modeling protein binding and elution over a chromatographic surface probed by surface plasmon resonance.

Surface plasmon resonance (SPR) spectroscopy is used as a scaled-down, analytical, pseudo-chromatography tool for analyzing protein binding and elution over an ion-exchange surface under cyclic sorption conditions. A micrometric-scale adsorption surface was produced by immobilizing a typical ion exchange ligand--diethylaminoethyl (DEAE)--onto commercially available planar gold sensor chip surfaces pre-derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid with known density. An explicit mathematical formulation is provided for the deconvolution and interpretation of the SPR sensorgrams. An adsorption rate model is proposed to describe the SPR sensorgrams for bovine serum albumin, used here as model protein, when the DEAE surface is subjected to a cyclic series of binding and elution steps. Overall, we demonstrate that the adsorption rate model is capable of quantitatively describing BSA binding and elution for protein titers from dilute conditions up to overloaded conditions and a broad range of salt concentrations.

Virus production for clinical gene therapy.

Gene therapy is becoming increasingly relevant for the treatment of prominent human diseases. Viral vectors are currently used in more than 50% of the gene therapy clinical trials, most of them aimed at cancer diseases. Clearly, the increasing needs of high-quality viral preparations require the elimination of process bottlenecks, streamlining the development of a viral vector into a real-world clinical tool. Virus production for clinical gene therapy can be a limiting step because many virus generation protocols rely on labor-intensive, bench-scale methods; robust, cost-effective strategies for the delivery of clinical-grade viruses are thus essential for the future of gene therapy. A comprehensive picture of key aspects on the integration of upstream and downstream processing is addressed in this chapter, by describing the case study of recombinant budded baculoviruses for gene therapy; scalable methods are described in detail as well as mandatory characterization techniques for a proper and complete quality assessment of the viral vectors.