MAR elements as tools to increase protein production by CHO cells

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection, analysis and characterization of the numerous clones required to identify one with the desired characteristics. Various boundary elements, matrix attachment regions, and locus control regions were screened for for their ability to augment the expression of heterologous genes in CHO and other cells. The 5'-matrix-attachment region (MAR) of the chicken lysozyme gene was found to significantly increase stable gene expression, in culture dishes and in bioreactors. These MAR elements can be easily combined with various existing expression systems, as they can be added in trans (i.e. on a separate plasmid) in co-transfections with previously constructed expression vectors. Using cell population analysis, we found that the use of the MAR increases the proportion of high-producing CHO cell clones, thus reducing the number of cell lines that need to be screened while increasing maximal productivity. Random cDNA cloning and sequencing indicated that over 12% of the ESTs correspond to the transgene. Thus, productivity is no longer limited by transcriptional events in such MAR-containing cell lines. The identification of small and more convenient active MAR portions will also be summarized. Finally, we will show examples of how MAR elements can be combined with short term expression to increase the simultaneous synthesis of many proteins in parallel by CHO cells. Overall, we conclude that the MAR sequence is a versatile tool to increase protein expression in short and long term production processes.

Pathways for the synthesis of polyesters in plants; cutin, suberin, and polyhydroxyalkanoates

Plants naturally produce the lipid-derived polyester cutin, which is found in the plant cuticle that is deposited at the outermost extracellular matrix of the epidermis covering nearly all aboveground tissues. Being at the interface between the cell and the external environment, cutin and the cuticle play important roles in the protection of plants from several stresses. A number of enzymes involved in the synthesis of cutin monomers have recently been identified, including several P450s and one acyl-CoA synthetase, thus representing the first steps toward the understanding of polyester formation and, potentially, polyester engineering to improve the tolerance of plants to stresses, such as drought, and for industrial applications. However, numerous processes underlying cutin synthesis, such as a controlled polymerization, still remain elusive. Suberin is a second polyester found in the extracellular matrix, most often synthesized in root tissues and during secondary growth. Similar to cutin, the function of suberin is to seal off the respective tissue to inhibit water loss and contribute to resistance to pathogen attack. Being the main constituent of cork, suberin is a plant polyester that has already been industrially exploited. Genetic engineering may be worth exploring in order to change the polyester properties for either different applications or to increase cork production in other species. Polyhydroxyalkanoates (PHAs) are attractive polyesters of 3-hydroxyacids because of their properties as bioplastics and elastomers. Although PHAs are naturally found in a wide variety of bacteria, biotechnology has aimed at producing these polymers in plants as a source of cheap and renewable biodegradable plastics. Synthesis of PHA containing various monomers has been demonstrated in the cytosol, plastids, and peroxisomes of plants. Several biochemical pathways have been modified in order to achieve this, including the isoprenoid pathway, the fatty acid biosynthetic pathway, and the fatty acid β-oxidation pathway. PHA synthesis has been demonstrated in a number of plants, including monocots and dicots, and up to 40% PHA per gram dry weight has been demonstrated in Arabidopsis thaliana. Despite some successes, production of PHA in crop plants remains a challenging project. PHA synthesis at high level in vegetative tissues, such as leaves, is associated with chlorosis and reduced growth. The challenge for the future is to succeed in synthesis of PHA copolymers with a narrow range of monomer compositions, at levels that do not compromise plant productivity. This goal will undoubtedly require a deeper understanding of plant biochemical pathways and how carbon fluxes through these pathways can be manipulated, areas where plant "omics" can bring very valuable contributions.

Determining the activity of Pu-241 by liquid scintillation counting

Liquid scintillation counting (LSC) is one of the most widely used methods for determining the activity of 241Pu. One of the main challenges of this counting method is the efficiency calibration of the system for the low beta energies of 241Pu (Emax = 20.8 keV). In this paper we compare the two most frequently used methods, the CIEMAT/NIST efficiency tracing (CNET) method and the experimental quench correction curve method. Both methods proved to be reliable, and agree within their uncertainties, for the expected quenching conditions of the sources.

Erkrankungen durch verotoxinbildende Escherichia coli beim Menschen [Verotoxin-producing Escherichia coli in human disease]

Les colibacilles producteurs de vérotoxines sont impliqués dans la pathogénie de syndromes diarrhéiques et dans celle de certains syndromes hémolytiques et urémiques. Le syndrome diarrhéique est caractérisé par l'apparition soudaine de douleurs abdominales sévères à type de crampes, suivies d'une diarrhée aqueuse qui, ensuite, devient sanglante. Les diarrhées peuvent être accompagnées de vomissements et d'une fièvre modérée. La période d'incubation varie entre 3 et 9 jours. Le syndrome hémolytique et urémique, la première cause d'insuffisance rénale aiguë du nourrisson et de l'enfant, est caractérisé par une triade typique: anémie hémolytique microangiopathique avec thrombocytopénie et insuffisance rénale glomérulonéphritique aiguë. La cause du syndrome hémolytique et urémique avec diarrhées prodromique a été attribuée aux vérotoxines. Les toxines produites par les colibacilles seraient à l'origine de lésions vasculaires endothéliales prédominant au niveau rénal et induisant le syndrome hémolytique et urémique.

Nucleic acid-containing amyloid fibrils potently induce type I interferon and stimulate systemic autoimmunity.

The immunopathophysiologic development of systemic autoimmunity involves numerous factors through complex mechanisms that are not fully understood. In systemic lupus erythematosus, type I IFN (IFN-I) produced by plasmacytoid dendritic cells (pDCs) critically promotes the autoimmunity through its pleiotropic effects on immune cells. However, the host-derived factors that enable abnormal IFN-I production and initial immune tolerance breakdown are largely unknown. Previously, we found that amyloid precursor proteins form amyloid fibrils in the presence of nucleic acids. Here we report that nucleic acid-containing amyloid fibrils can potently activate pDCs and enable IFN-I production in response to self-DNA, self-RNA, and dead cell debris. pDCs can take up DNA-containing amyloid fibrils, which are retained in the early endosomes to activate TLR9, leading to high IFNα/β production. In mice treated with DNA-containing amyloid fibrils, a rapid IFN response correlated with pDC infiltration and activation. Immunization of nonautoimmune mice with DNA-containing amyloid fibrils induced antinuclear serology against a panel of self-antigens. The mice exhibited positive proteinuria and deposited antibodies in their kidneys. Intriguingly, pDC depletion obstructed IFN-I response and selectively abolished autoantibody generation. Our study reveals an innate immune function of nucleic acid-containing amyloid fibrils and provides a potential link between compromised protein homeostasis and autoimmunity via a pDC-IFN axis.

Cell disposition of raltegravir and newer antiretrovirals in HIV-infected patients: high inter-individual variability in raltegravir cellular penetration.

Objectives The site of pharmacological activity of raltegravir is intracellular. Our aim was to determine the extent of raltegravir cellular penetration and whether raltegravir total plasma concentration (C(tot)) predicts cellular concentration (C(cell)). Methods Open-label, prospective, pharmacokinetic study on HIV-infected patients on a stable raltegravir-containing regimen. Plasma and peripheral blood mononuclear cells were simultaneously collected during a 12 h dosing interval after drug intake. C(tot) and C(cell) of raltegravir, darunavir, etravirine, maraviroc and ritonavir were measured by liquid chromatography coupled to tandem mass spectrometry after protein precipitation. Longitudinal mixed effects analysis was applied to the C(cell)/C(tot) ratio. Results Ten HIV-infected patients were included. The geometric mean (GM) raltegravir total plasma maximum concentration (C(max)), minimum concentration (C(min)) and area under the time-concentration curve from 0-12 h (AUC(0-12)) were 1068 ng/mL, 51.1 ng/mL and 4171 ng·h/mL, respectively. GM raltegravir cellular C(max), C(min) and AUC(0-12) were 27.5 ng/mL, 2.9 ng/mL and 165 ng·h/mL, respectively. Raltegravir C(cell) corresponded to 5.3% of C(tot) measured simultaneously. Both concentrations fluctuate in parallel, with C(cell)/C(tot) ratios remaining fairly constant for each patient without a significant time-related trend over the dosing interval. The AUC(cell)/AUC(tot) GM ratios for raltegravir, darunavir and etravirine were 0.039, 0.14 and 1.55, respectively. Conclusions Raltegravir C(cell) correlated with C(tot) (r = 0.86). Raltegravir penetration into cells is low overall (∼5% of plasma levels), with distinct raltegravir cellular penetration varying by as much as 15-fold between patients. The importance of this finding in the context of development of resistance to integrase inhibitors needs to be further investigated.

The region of mouse mammary tumor virus DNA containing the long terminal repeat includes a long coding sequence and signals for hormonally regulated transcription.

Starting from a biologically active recombinant DNA clone of exogenous unintegrated GR mouse mammary tumor virus, we have generated three subclones of PstI fragments of 1.45, 1.1, and 2.0 kb in the plasmid vector PBR322. The nucleotide sequence has been determined for the clone of 1.45 kb which includes almost the complete region of the long terminal repeat (LTR) plus an adjacent stretch of unique sequence DNA. A short region of the 2.0 kb clone, containing the beginning of the LTR, has also been sequenced. Starting with the A of an initiation codon outside the LTR, we detected an open reading frame of 960 nucleotides, potentially coding for a protein of 320 amino acids (36K). Two hundred nucleotides downstream from the termination codon, and approximately 25 nucleotides upstream from the presumptive initiation site of viral RNA synthesis, we found a promoter-like sequence. The sequence AGTAAA was detected approximately 15-20 nucleotides upstream from the 3' end of virion RNA and probably serves as a polyadenylation signal. The 1.45 kb PstI fragment has been transfected into Ltk- cells together with a plasmid containing the thymidine kinase gene of herpes simplex virus. The virus-specific RNA synthesis detected in a Tk+ cell clone was strongly stimulated by the addition of dexamethasone.

Magnetic and in vitro heating properties of implants formed in situ from injectable formulations and containing superparamagnetic iron oxide nanoparticles (SPIONs) embedded in silica microparticles for magnetically induced local hyperthermia

The biological and therapeutic responses to hyperthermia, when it is envisaged as an anti-tumor treatment modality, are complex and variable. Heat delivery plays a critical role and is counteracted by more or less efficient body cooling, which is largely mediated by blood flow. In the case of magnetically mediated modality, the delivery of the magnetic particles, most often superparamagnetic iron oxide nanoparticles (SPIONs), is also critically involved. We focus here on the magnetic characterization of two injectable formulations able to gel in situ and entrap silica microparticles embedding SPIONs. These formulations have previously shown suitable syringeability and intratumoral distribution in vivo. The first formulation is based on alginate, and the second on a poly(ethylene-co-vinyl alcohol) (EVAL). Here we investigated the magnetic properties and heating capacities in an alternating magnetic field (141 kHz, 12 mT) for implants with increasing concentrations of magnetic microparticles. We found that the magnetic properties of the magnetic microparticles were preserved using the formulation and in the wet implant at 37 degrees C, as in vivo. Using two orthogonal methods, a common SLP (20 Wg(-1)) was found after weighting by magnetic microparticle fraction, suggesting that both formulations are able to properly carry the magnetic microparticles in situ while preserving their magnetic properties and heating capacities. (C) 2010 Elsevier B.V. All rights reserved.

Extended bottom-up proteomics with secreted aspartic protease Sap9.

We investigate the benefits and experimental feasibility of approaches enabling the shift from short (1.7kDa on average) peptides in bottom-up proteomics to about twice longer (~3.2kDa on average) peptides in the so-called extended bottom-up proteomics. Candida albicans secreted aspartic protease Sap9 has been selected for evaluation as an extended bottom-up proteomic-grade enzyme due to its suggested dibasic cleavage specificity and ease of production. We report the extensive characterization of Sap9 specificity and selectivity revealing that protein cleavage by Sap9 most often occurs in the vicinity of proximal basic amino acids, and in select cases also at basic and hydrophobic residues. Sap9 is found to cleave a large variety of proteins in a relatively short, ~1h, period of time and it is efficient in a broad pH range, including slightly acidic, e. g., pH5.5, conditions. Importantly, the resulting peptide mixtures contain representative peptides primarily in the target 3-7kDa range. The utility and advantages of this enzyme in routine analysis of protein mixtures are demonstrated and the limitations are discussed. Overall, Sap9 has a potential to become an enzyme of choice in an extended bottom-up proteomics, which is technically ready to complement the traditional bottom-up proteomics for improved targeted protein structural analysis and expanded proteome coverage. BIOLOGICAL SIGNIFICANCE: Advances in biological applications of mass spectrometry-based bottom-up proteomics are oftentimes limited by the extreme complexity of biological samples, e.g., proteomes or protein complexes. One of the reasons for it is in the complexity of the mixtures of enzymatically (most often using trypsin) produced short (<3kDa) peptides, which may exceed the analytical capabilities of liquid chromatography and mass spectrometry. Information on localization of protein modifications may also be affected by the small size of typically produced peptides. On the other hand, advances in high-resolution mass spectrometry and liquid chromatography have created an intriguing opportunity of improving proteome analysis by gradually increasing the size of enzymatically-derived peptides in MS-based bottom-up proteomics. Bioinformatics has already confirmed the envisioned advantages of such approach. The remaining bottle-neck is an enzyme that could produce longer peptides. Here, we report on the characterization of a possible candidate enzyme, Sap9, which may be considered for producing longer, e.g., 3-7kDa, peptides and lead to a development of extended bottom-up proteomics.

Generic approach for the sensitive absolute quantification of large undigested peptides in plasma using a particular liquid chromatography-mass spectrometry setup.

A generic LC-MS approach for the absolute quantification of undigested peptides in plasma at mid-picomolar levels is described. Nine human peptides namely, brain natriuretic peptide (BNP), substance P (SubP), parathyroid hormone 1-34 (PTH), C-peptide, orexines A and B (Orex-A and -B), oxytocin (Oxy), gonadoliberin-1 (gonadothropin releasing-hormone or luteinizing hormone-releasing hormone, LHRH) and α-melanotropin (α-MSH) were targeted. Plasma samples were extracted via a 2-step procedure: protein precipitation using 1vol of acetonitrile followed by ultrafiltration of supernatants on membranes with a MW cut-off of 30 kDa. By applying a specific LC-MS setup, large volumes of filtrates (e.g., 2×750 μL) were injected and the peptides were trapped on a 1mm i.d.×10 mm length C8 column using a 10× on-line dilution. Then, the peptides were back-flushed and a second on-line dilution (2×) was applied during the transfer step. The refocalized peptides were resolved on a 0.3mm i.d. C18 analytical column. Extraction recovery, matrix effect and limits of detection were evaluated. Our comprehensive protocol demonstrates a simple and efficient sample preparation procedure followed by the analysis of peptides with limits of detection in the mid-picomolar range. This generic approach can be applied for the determination of most therapeutic peptides and possibly for endogenous peptides with latest state-of-the-art instruments.

Long-term ex vivo expansion of human fetal liver primitive haematopoietic progenitor cells in stroma-free cultures

Successful expansion of haematopoietic cells in ex vivo cultures will have important applications in transplantation, gene therapy, immunotherapy and potentially also in the production of non-haematopoietic cell types. Haematopoietic stem cells (HSC), with their capacity to both self-renew and differentiate into all blood lineages, represent the ideal target for expansion protocols. However, human HSC are rare, poorly characterized phenotypically and genotypically, and difficult to test functionally. Defining optimal culture parameters for ex vivo expansion has been a major challenge. We devised a simple and reproducible stroma-free liquid culture system enabling long-term expansion of putative haematopoietic progenitors contained within frozen human fetal liver (FL) crude cell suspensions. Starting from a small number of total nucleated cells, a massive haematopoietic cell expansion, reaching > 1013-fold the input cell number after approximately 300 d of culture, was consistently achieved. Cells with a primitive phenotype were present throughout the culture and also underwent a continuous expansion. Moreover, the capacity for multilineage lymphomyeloid differentiation, as well as the recloning capacity of primitive myeloid progenitors, was maintained in culture. With its better proliferative potential as compared with adult sources, FL represents a promising alternative source of HSC and the culture system described here should be useful for clinical applications.

Advantages of extended bottom-up proteomics using sap9 for analysis of monoclonal antibodies.

Despite the recent advances in structural analysis of monoclonal antibodies with bottom-up, middle-down, and top-down mass spectrometry (MS), further improvements in analysis accuracy, depth, and speed are needed. The remaining challenges include quantitatively accurate assignment of post-translational modifications, reduction of artifacts introduced during sample preparation, increased sequence coverage per liquid chromatography (LC) MS experiment, and ability to extend the detailed characterization to simple antibody cocktails and more complex antibody mixtures. Here, we evaluate the recently introduced extended bottom-up proteomics (eBUP) approach based on proteolysis with secreted aspartic protease 9, Sap9, for analysis of monoclonal antibodies. Key findings of the Sap9-based proteomics analysis of a single antibody include: (i) extensive antibody sequence coverage with up to 100% for the light chain and up to 99-100% for the heavy chain in a single LC-MS run; (ii) connectivity of complementarity-determining regions (CDRs) via Sap9-produced large proteolytic peptides (3.4 kDa on average) containing up to two CDRs per peptide; (iii) reduced artifact introduction (e. g., deamidation) during proteolysis with Sap9 compared to conventional bottom-up proteomics workflows. The analysis of a mixture of six antibodies via Sap9-based eBUP produced comparable results. Due to the reasons specified above, Sap9-produced proteolytic peptides improve the identification confidence of antibodies from the mixtures compared to conventional bottom-up proteomics dealing with shorter proteolytic peptides.

Polyhydroxyalkanoate synthesis in transgenic plants as a new tool to study carbon flow through beta-oxidation.

Transgenic plants producing peroxisomal polyhydroxy- alkanoate (PHA) from intermediates of fatty acid degradation were used to study carbon flow through the beta-oxidation cycle. Growth of transgenic plants in media containing fatty acids conjugated to Tween detergents resulted in an increased accumulation of PHA and incorporation into the polyester of monomers derived from the beta-oxidation of these fatty acids. Tween-laurate was a stronger inducer of beta-oxidation, as measured by acyl-CoA oxidase activity, and a more potent modulator of PHA quantity and monomer composition than Tween-oleate. Plants co-expressing a peroxisomal PHA synthase with a capryl-acyl carrier protein thioesterase from Cuphea lanceolata produced eightfold more PHA compared to plants expressing only the PHA synthase. PHA produced in double transgenic plants contained mainly saturated monomers ranging from 6 to 10 carbons, indicating an enhanced flow of capric acid towards beta-oxidation. Together, these results support the hypothesis that plant cells have mechanisms which sense levels of free or esterified unusual fatty acids, resulting in changes in the activity of the beta-oxidation cycle as well as removal and degradation of these unusual fatty acids through beta-oxidation. Such enhanced flow of fatty acids through beta-oxidation can be utilized to modulate the amount and composition of PHA produced in transgenic plants. Furthermore, synthesis of PHAs in plants can be used as a new tool to study the quality and relative quantity of the carbon flow through beta-oxidation as well as to analyse the degradation pathway of unusual fatty acids.

Quantification of typical antipsychotics in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A selective and sensitive method was developed for the simultaneous quantification of seven typical antipsychotic drugs (cis-chlorprothixene, flupentixol, haloperidol, levomepromazine, pipamperone, promazine and zuclopenthixol) in human plasma. Ultra-high performance liquid chromatography (UHPLC) was used for complete separation of the compounds in less than 4.5min on an Acquity UPLC BEH C18 column (2.1mm×50mm; 1.7μm), with a gradient elution of ammonium formate buffer pH 4.0 and acetonitrile at a flow rate of 400μl/min. Detection was performed on a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray ionization interface. A simple protein precipitation procedure with acetonitrile was used for sample preparation. Thanks to the use of stable isotope-labeled internal standards for all analytes, internal standard-normalized matrix effects were in the range of 92-108%. The method was fully validated to cover large concentration ranges of 0.2-90ng/ml for haloperidol, 0.5-90ng/ml for flupentixol, 1-450ng/ml for levomepromazine, promazine and zuclopenthixol and 2-900ng/ml for cis-chlorprothixene and pipamperone. Trueness (89.1-114.8%), repeatability (1.8-9.9%), intermediate precision (1.9-16.3%) and accuracy profiles (<30%) were in accordance with the latest international recommendations. The method was successfully used in our laboratory for routine quantification of more than 500 patient plasma samples for therapeutic drug monitoring. To the best of our knowledge, this is the first UHPLC-MS/MS method for the quantification of the studied drugs with a sample preparation based on protein precipitation.