On-line monitoring of responses to nutrient feed additions by multi-frequency permittivity measurements in fed-batch cultivations of CHO cells

Changes in the nutrient availability of mammalian cell cultures are reflected in the β-dispersion parameter characteristic frequency (f _C ) and the on-line dual frequency permittivity signal. Multi-frequency permittivity measurements were therefore evaluated in fed-batch cultivations of two different CHO cell lines. Similar responses to nutrient depletions and discontinuous feed additions were monitored in different cultivation phases and experimental setups. Sudden increases in permittivity and f _C occurred when feed additions were conducted. A constant or declining permittivity value in combination with a decrease in f _C indicated nutrient limitations. f _C correlated well with changes in oxygen uptake rate when cell diameter remained constant, indicating that metabolic activity is reflected in the value of f _C . When significant cell size changes occurred during the cultivations, the analysis of the β-dispersion parameters was rendered complex. For the application of our findings in other systems it will be hence required to conduct additional off-line measurements. Based on these results, it is hypothesized that multi-frequency permittivity measurements can give information on the intracellular or physiological state in fed-batch mode. Similar observations were made when using different cell lines and feeding strategies, indicating that the findings are transferable to other cell lines and systems. The results should lead to an improved understanding of routine fed-batch processes. Additional studies are, however, required to explore how these observations can be used for fed-batch process development and optimization.     

Detecting un-authorized genetically modified organisms (GMOs) and derived materials

Genetically modified plants, in the following referred to as genetically modified organisms or GMOs, have been commercially grown for almost two decades. In 2010 approximately 10% of the total global crop acreage was planted with GMOs (James, 2011). More than 30 countries have been growing commercial GMOs, and many more have performed field trials. Although the majority of commercial GMOs both in terms of acreage and specific events belong to the four species: soybean, maize, cotton and rapeseed, there are another 20 + species where GMOs are commercialized or in the pipeline for commercialization. The number of GMOs cultivated in field trials or for commercial production has constantly increased during this time period. So have the number of species, the number of countries involved, the diversity of novel (added) genetic elements and the global trade. All of these factors contribute to the increasing complexity of detecting and correctly identifying GMO derived material. Many jurisdictions, including the European Union (EU), legally distinguish between authorized (and therefore legal) and un-authorized (and therefore illegal) GMOs. Information about the developments, field trials, authorizations, cultivation, trade and observations made in the official GMO control laboratories in different countries around the world is often limited, despite several attempts such as the OECD BioTrack for voluntary dissemination of data. This lack of information inevitably makes it challenging to detect and identify GMOs, especially the un-authorized GMOs. The present paper reviews the state of the art technologies and approaches in light of coverage, practicability, sensitivity and limitations. Emphasis is put on exemplifying practical detection of un-authorized GMOs. Although this paper has a European (EU) bias when examples are given, the contents have global relevance.     

Determination of T-2 toxin,HT-2 toxin,and three other type A trichothecenes in layer feed by high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS)—comparison of two sample preparation methods

A sensitive method for the simultaneous determination of T-2 toxin, HT-2 toxin, neosolaniol, T-2 triol, and T-2 tetraol in layer feed using high-performance liquid chromatography coupled to triple quadrupole mass spectrometry in the positive ionization mode (LC-ESI-MS/MS) is described. Two fast and easy clean-up methods—with BondElut Mycotoxin and MycoSep 227 columns, respectively—were tested. The separation of the toxins was conducted on a Pursuit XRs Ultra 2.8 HPLC column using 0.13 mM ammonium acetate as eluent A and methanol as eluent B. Detection of the mycotoxins was carried out in the multiple reaction monitoring (MRM) mode using ammonium adducts as precursor ions. Quantification of all analytes was performed with d₃-T-2 toxin as an internal standard. The clean-up method with MycoSep 227 columns gave slightly better results for layer feed compared to the method using BondElut Mycotoxin columns (MycoSep 227: recovery between 50 and 63 %, BondElut Mycotoxin: recovery between 32 and 67 %) and was therefore chosen as the final method. The limits of detection ranged between 0.9 and 7.5 ng/g depending on the mycotoxin. The method was developed for the analysis of layer feed used at carry-over experiments with T-2 toxin in laying hens. For carry-over experiments, it is necessary that the method includes not only T-2 toxin but also the potential metabolites in animal tissues HT-2 toxin, neosolaniol, T-2 triol, and T-2 tetraol which could naturally occur in cereals used as feed stuff as well.     

A β-Lactam Antibiotic Dampens Excitotoxic Inflammatory CNS Damage in a Mouse Model of Multiple Sclerosis

In multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE), impairment of glial “Excitatory Amino Acid Transporters” (EAATs) together with an excess glutamate-release by invading immune cells causes excitotoxic damage of the central nervous system (CNS). In order to identify pathways to dampen excitotoxic inflammatory CNS damage, we assessed the effects of a β-lactam antibiotic, ceftriaxone, reported to enhance expression of glial EAAT2, in “Myelin Oligodendrocyte Glycoprotein” (MOG)-induced EAE. Ceftriaxone profoundly ameliorated the clinical course of murine MOG-induced EAE both under preventive and therapeutic regimens. However, ceftriaxone had impact neither on EAAT2 protein expression levels in several brain areas, nor on the radioactive glutamate uptake capacity in a mixed primary glial cell-culture and the glutamate-induced uptake currents in a mammalian cell line mediated by EAAT2. Moreover, the clinical effect of ceftriaxone was preserved in the presence of the EAAT2-specific transport inhibitor, dihydrokainate, while dihydrokainate alone caused an aggravated EAE course. This demonstrates the need for sufficient glial glutamate uptake upon an excitotoxic autoimmune inflammatory challenge of the CNS and a molecular target of ceftriaxone other than the glutamate transporter. Ceftriaxone treatment indirectly hampered T cell proliferation and proinflammatory INFγ and IL17 secretion through modulation of myelin-antigen presentation by antigen-presenting cells (APCs) e.g. dendritic cells (DCs) and reduced T cell migration into the CNS in vivo. Taken together, we demonstrate, that a β-lactam antibiotic attenuates disease course and severity in a model of autoimmune CNS inflammation. The mechanisms are reduction of T cell activation by modulation of cellular antigen-presentation and impairment of antigen-specific T cell migration into the CNS rather than or modulation of central glutamate homeostasis.     

Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects

The E3 ubiquitin ligase PARK2 and the mitochondrial protein kinase PINK1 are required for the initiation of mitochondrial damage-induced mitophagy. Together, PARK2 and PINK1 generate a phospho-ubiquitin signal on outer mitochondrial membrane proteins that triggers recruitment of the autophagy machinery. This paper describes the detection of a defined 500-kDa phospho-ubiquitin-rich PARK2 complex that accumulates on mitochondria upon treatment with the membrane uncoupler CCCP. Formation of this complex is dependent on the presence of PINK1 and is absent in mutant forms of PARK2, whereby mitophagy is also arrested. These results signify a functional signaling complex that is essential for the progression of mitophagy. The visualization of the PARK2 signaling complex represents a novel marker for this critical step in mitophagy and can be used to monitor mitophagy progression in PARK2 mutants and to uncover additional upstream factors required for PARK2-mediated mitophagy signaling.     

Protein kinase C remains functionally active during TPA induced neuronal differentiation of SH-SY5Y human neuroblastoma cells.

SH-SY5Y human neuroblastoma cells can be induced to differentiate into a neuronal phenotype by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In other cell systems, TPA treatment frequently leads to down-regulation of protein kinase C (PKC). However, we now report that TPA-treated and non-treated SH-SY5Y cells express PKC-alpha, but not PKC-beta and PKC-gamma, mRNA. Furthermore, only a slight down-regulation of the PKC-alpha protein could be seen during prolonged treatment with 16 nM TPA, the concentration giving optimal differentiation. In contrast, a higher concentration of TPA (1.6 microM) results in a poor neuronal differentiation and a complete down-regulation of PKC-alpha. PKC-alpha was rapidly translocated to the particulate fraction and remained membrane bound for at least 4 days during treatment with 16 nM TPA. In such cells a sustained increased level of the phosphorylated form of a 80,000 Dalton PKC-substrate was found. In addition to this sustained augmented phosphorylation, administration of fresh TPA at day 4 caused a small but reproducible further increased level of phosphorylated substrate. When the PKC activity was measured by the histone phosphorylation assay a substantial fraction of the initial enzyme activity could still be detected after 4 days of TPA treatment. Taken together, the data demonstrate that PKC remains functionally active during TPA induced differentiation of SH-SY5Y cells, which may suggest a continuous role for the enzyme during the differentiation process.     

Neoascarophis macrouri n. sp. (Nematoda: Cystidicolidae) from the stomach of Macrourus berglax (Macrouridae) in the eastern Greenland Sea

A new species of parasitic nematode, Neoascarophis macrouri n. sp. (Cystidicolidae), is described from the stomach and stomach wall of the marine deep-water fish Macrourus berglax (onion-eye grenadier) in the eastern Greenland Sea (North Atlantic Ocean). The new species, studied using both light and scanning electron microscopy, is characterised mainly by the location of the vulva near the posterior end of the body (a short distance anterior to the anus), non-filamented eggs, the structure of the mouth, a short vestibule and the length of the spicules (567–615 and 144–156 μm). Metabronema insulanum Solov’eva, 1991 is transferred to Neoascarophis as N. insulana (Solov’eva, 1991) n. comb.     

Ion-specific Effects on Prion Nucleation and Strain Formation

Ordered, fibrous, self-seeding aggregates of misfolded proteins known as amyloids are associated with important diseases in mammals and control phenotypic traits in fungi. A given protein may adopt multiple amyloid conformations, known as variants or strains, each of which leads to a distinct disease pattern or phenotype. Here, we study the effect of Hofmeister ions on amyloid nucleation and strain generation by the prion domain-containing fragment (Sup35NM) of a yeast protein Sup35p. Strongly hydrated anions (kosmotropes) initiate nucleation quickly and cause rapid fiber elongation, whereas poorly hydrated anions (chaotropes) delay nucleation and mildly affect the elongation rate. For the first time, we demonstrate that kosmotropes favor formation of amyloid strains that are characterized by lower thermostability and higher frangibility in vitro and stronger phenotypic and proliferation patterns effectively in vivo as compared with amyloids formed in chaotropes. These phenomena point to inherent differences in the biochemistry of Hofmeister ions. Our work shows that the ionic composition of a solution not only influences the kinetics of amyloid nucleation but also determines the amyloid strain that is preferentially formed.     

Revised Vertebral Count in the “Longest-Necked Vertebrate” Elasmosaurus platyurus Cope 1868, and Clarification of the Cervical-Dorsal Transition in Plesiosauria

Elasmosaurid plesiosaurians are renowned for their immensely long necks, and indeed, possessed the highest number of cervical vertebrae for any known vertebrate. Historically, the largest count has been attributed to the iconic Elasmosaurus platyurus from the Late Cretaceous of Kansas, but estimates for the total neck series in this taxon have varied between published reports. Accurately determining the number of vertebral centra vis-à-vis the maximum length of the neck in plesiosaurians has significant implications for phylogenetic character designations, as well as the inconsistent terminology applied to some osteological structures. With these issues in mind, we reassessed the holotype of E. platyurus as a model for standardizing the debated cervical-dorsal transition in plesiosaurians, and during this procedure, documented a “lost” cervical centrum. Our revision also advocates retention of the term “pectorals” to describe the usually three or more distinctive vertebrae close to the cranial margin of the forelimb girdle that bear a functional rib facet transected by the neurocentral suture, and thus conjointly formed by both the parapophysis on the centrum body and diapophysis from the neural arch (irrespective of rib length). This morphology is unambiguously distinguishable from standard cervicals, in which the functional rib facet is borne exclusively on the centrum, and dorsals in which the rib articulation is situated above the neurocentral suture and functionally borne only by the transverse process of the neural arch. Given these easily distinguishable definitions, the maximum number of neck vertebrae preserved in E. platyurus is 72; this is only three vertebrae shorter than the recently described Albertonectes, which together with E. platyurus constitute the “longest necked” animals ever to have lived.