Identification and Monitoring of Host Cell Proteins by Mass Spectrometry Combined with High Performance Immunochemistry Testing

Biotherapeutics are often produced in non-human host cells like Escherichia coli, yeast, and various mammalian cell lines. A major focus of any therapeutic protein purification process is to reduce host cell proteins to an acceptable low level. In this study, various E. coli host cell proteins were identified at different purifications steps by HPLC fractionation, SDS-PAGE analysis, and tryptic peptide mapping combined with online liquid chromatography mass spectrometry (LC-MS). However, no host cell proteins could be verified by direct LC-MS analysis of final drug substance material. In contrast, the application of affinity enrichment chromatography prior to comprehensive LC-MS was adequate to identify several low abundant host cell proteins at the final drug substance level. Bacterial alkaline phosphatase (BAP) was identified as being the most abundant host cell protein at several purification steps. Thus, we firstly established two different assays for enzymatic and immunological BAP monitoring using the cobas® technology. By using this strategy we were able to demonstrate an almost complete removal of BAP enzymatic activity by the established therapeutic protein purification process. In summary, the impact of fermentation, purification, and formulation conditions on host cell protein removal and biological activity can be conducted by monitoring process-specific host cell proteins in a GMP-compatible and high-throughput (> 1000 samples/day) manner.     

Genetic Variability of Microcystin Biosynthesis Genes in Planktothrix as Elucidated from Samples Preserved by Heat Desiccation during Three Decades

Historic samples of phytoplankton can provide information on the abundance of the toxigenic genotypes of cyanobacteria in dependence on increased or decreased eutrophication. The analysis of a time-series from preserved phytoplankton samples by quantitative PCR (qPCR) extends observation periods considerably. The analysis of DNA from heat-desiccated samples by qPCR can be aggravated by point substitutions or the fragmentation of DNA introduced by the high temperature. In this study, we analyzed whether the heat desiccation of the cellular material of the cyanobacterium Planktothrix sp. introduced potential errors to the template DNA that is used for qPCR within (i) 16S rDNA and phycocyanin genes and (ii) the mcyA gene indicative of the incorporation of either dehydrobutyrine (Dhb) or N-methyl-dehydroalanine (Mdha) in position 7, and (ii) the mcyB gene, which is indicative of homotyrosine (Hty) in position 2 of the microcystin (MC) molecule. Due to high temperature desiccation, the deterioration of the DNA template quality was rather due to fragmentation than due to nucleotide substitutions. By using the heat-desiccated samples of Lake Zürich, Switzerland the abundance of the Dhb, Mdha and Hty genotypes was determined during three decades (1977-2008). Despite major changes in the trophic state of the lake resulting in a major increase of the total Planktothrix population density, the proportion of these genotypes encoding the synthesis of different MC congeners showed high stability. Nevertheless, a decline of the most abundant mcyA genotype indicative of the synthesis of Dhb in position 7 of the MC molecule was observed. This decline could be related to the gradual incline in the proportion of a mutant genotype carrying a 1.8kbp deletion of this gene region. The increase of this mcyA (Dhb) gene deletion mutant has been minor so far, however, and likely did not affect the overall toxicity of the population.     

Clinical and functional characterisation of the combined respiratory chain defect in two sisters due to autosomal recessive mutations in MTFMT

Exome sequencing identified compound heterozygous mutations in the recently discovered mitochondrial methionyl-tRNA formyltransferase (MTFMT) gene in two sisters with mild Leigh syndrome and combined respiratory chain deficiency. The mutations lead to undetectable levels of the MTFMT protein. Blue native polyacrylamide gel electrophoresis showed decreased complexes I and IV, and additional products stained with complex V antibodies, however the overall steady state level of mt-tRNA^Met was normal. Our data illustrate that exome sequencing is an excellent diagnostic tool, and its value in clinical medicine is enormous, however it can only be optimally exploited if combined with detailed phenotyping and functional studies.     

Lipofection and nucleofection of substrate plasmid can generate widely different readings of DNA end-joining efficiency in different cell lines

In vivo plasmid end-joining assays are valuable tools for dissecting important qualitative and quantitative aspects of non-homologous end-joining (NHEJ) – a key mechanism for the repair of DNA double-strand breaks (DSBs) in higher eukaryotes. They enable the use of defined DNA ends as substrates for end-joining and the analysis by sequencing of the resulting junctions to identify the repair pathways engaged. Yet, plasmid assays have generated divergent results of end-joining capacity in the same DSB repair mutants when used under different conditions, which implies contributions from undefined and therefore uncontrolled parameters. To help standardize these assays, we searched for parameters underpinning these variations and identified transfection method as an important determinant. Here, we compare a lipid-based transfection method, lipofection, with an electroporation method, nucleofection, and find large, unanticipated and cell line-dependent differences in percent end-joining without recognizable trends. For example, in rodent cells, transfection using lipofection gives nearly WT end-joining in DNA-PKcs mutants and only mildly inhibited end-joining in Lig4 and Ku mutants. In contrast, transfection using nucleofection shows marked end-joining inhibition in all NHEJ mutants tested as compared to the WT. In human HCT116 cells, end-joining after nucleofection is strongly suppressed even in the WT and the differences to the mutants are small. After lipofection, in contrast, end-joining is high in WT cells and markedly suppressed in the mutants. We conclude that better understanding and control of the physicochemical/biological and analytical parameters underpinning these differences will be required to generate with plasmid assays results with quantitative power comparable to that of well-established methods of DSB analysis such as pulsed-field gel electrophoresis or γ-H2AX foci scoring. Until then, caution is needed in the interpretation of the results obtained – particularly with reference to pathway efficiency and residual damage – and confirmation of critical results with alternative transfection approaches is advisable.     

Advanced Glycation End Products in Infant Formulas Do Not Contribute to Insulin Resistance Associated with Their Consumption

Introduction

Infant formula-feeding is associated with reduced insulin sensitivity. In rodents and healthy humans, advanced glycation end product (AGE)-rich diets exert diabetogenic effects. In comparison with human breast-milk, infant formulas contain high amounts of AGEs. We assessed the role of AGEs in infant-formula-consumption-associated insulin resistance.

Methods

Total plasma levels of N^ε-(carboxymethyl)lysine (CML), AGEs-associated fluorescence (λ_ex = 370 nm/λ_em = 445 nm), soluble adhesion molecules, markers of micro- binflammation (hsCRP), oxidative stress (malondialdehyde, 8-isoprostanes) and leptinemia were determined, and correlated with insulin sensitivity in a cross-sectional study in 166 healthy term infants aged 3-to-14 months, subdivided according to feeding regimen (breast-milk- vs. infant formula-fed) and age (3-to-6-month-olds, 7-to-10-month-olds, and 11-to-14-month-old infants). Effects of the consumption of low- vs. high-CML-containing formulas were assessed. 36 infants aged 5.8±0.3 months were followed-up 7.5±0.3 months later.

Results

Cross-sectional study: 3-to-6-month-olds and 7-to-10-month-old formula-fed infants presented higher total plasma CML levels and AGEs-associated fluorescence (p<0.01, both), while only the 3-to-6-month-olds displayed lower insulin sensitivity (p<0.01) than their breast-milk-fed counterparts. 3-to-6-month-olds fed low-CML-containing formulas presented lower total plasma CML levels (p<0.01), but similar insulin sensitivity compared to those on high-CML-containing formulas. Markers of oxidative stress and inflammation, levels of leptin and adhesion molecules did not differ significantly between the groups. Follow-up study: at initial investigation, the breast-milk-consuming infants displayed lower total plasma CML levels (p<0.01) and AGEs-associated fluorescence (p<0.05), but higher insulin sensitivity (p<0.05) than the formulas-consuming infants. At follow-up, the groups did not differ significantly in either determined parameter.

Conclusions

In healthy term infants, high dietary load with CML does not play a pathophysiological role in the induction of infant formula-associated insulin resistance. Whether a high load of AGEs in early childhood affects postnatal programming remains to be elucidated.     

Sex-based differences in vigor and site preferences of Juniperus communis subsp. nana

Dioecious alpine juniper has been influenced by human impacts, management and climate changes; hence, its present populations are remnant fragments of its former distribution in central Europe. Complex environmental shifts have had fatal consequences for growth, reproduction and health of juniper and hence for its population structure. We asked the questions: 1) what was the population size structure, the sex ratio and the health status of individuals? 2) How were the population parameters linked with the environmental conditions and surrounding vegetation? The study area was close and above the upper forest limit in central Europe, the Hrubý Jeseník Mts. The parameters of each juniper individual and its environmental conditions were obtained, Ellenberg's indicator values and habitat categories were also determined. Proportions of sex categories, shrub size and environmental conditions were investigated to assess relationships between the population categories and environmental factors. We revealed linkage among the shrub size, health vigor, vegetation cover, a habitat and environmental factors. While there was equal ratio of females and males (1:1), small individuals of undetermined sex predominated that reducing the effective population size. Juniper health vigor was associated with surrounding vegetation cover. Individuals of undetermined sex were smaller than those of remaining sex categories and prefer specific habitats at higher elevation, underlining the effect of abiotic conditions on sex performance and ratio. The observed pattern, which was attributed to shift in land-use practices in the middle of 20th century and climate changes, could be improved by management. We recommended experimental local grazing and mowing accompanying by profound and continuing assessment of interaction among environmental factors and juniper performance.     

The importance of seasonal environmental factors in the foraging habitat selection of Alpine Ring Ouzels Turdus torquatus alpestris

Species inhabiting mountain ecosystems are expected to be particularly vulnerable to environmental change, yet information on their basic ecology is often lacking. Knowledge from field-based empirical studies remains essential to refine our understanding of the impact of current habitat alterations and for the consequential development of meaningful conservation management strategies. This study focuses on a poorly investigated and vulnerable mountain bird species in Europe, the Ring Ouzel Turdus torquatus. Our aim was to identify the species’ key ecological requirements during the crucial period of nestling provisioning in the context of environmental change. We radiotracked and observed Alpine Ring Ouzels in a high-density population, investigating their pattern of foraging habitat selection in 2015 and 2017, and evaluated the transferability of these results over a wider geographical range across the SW Swiss Alps. Foraging birds selected, consistently in space and time, short grass swards (< 10 cm) with interspersed patches of accessible and penetrable soils, at intermediate moisture levels (around 40–65% volumetric water content). In Alpine ecosystems, this microhabitat configuration is typically widespread during the spring snowmelt, but extremely seasonal, with a rapid decrease in its availability over the course of the breeding season. This underlines the high vulnerability of the Ring Ouzel to environmental change: an earlier snowmelt could generate a temporal mismatch between the peak of the breeding effort and optimal foraging conditions; however, abandoning grazing activities on semi-wooded Alpine pastures may further decrease foraging habitat suitability through taller and denser grass swards, and subsequent woody vegetation encroachment. This study provides a mechanistic appraisal of the challenges Ring Ouzels will face in the future, as well as initial guidelines for targeted habitat management within timberline ecotones.     

Natural mutations lead to enhanced proteasomal degradation of human Ncb5or,a novel flavoheme reductase

NADH cytochrome b₅ oxidoreductase (Ncb5or) protects β-cells against oxidative stress and lipotoxicity. The predominant phenotype of lean Ncb5or-null mouse is insulin-dependent diabetes due to β-cell death. This suggests the putative role of NCB5OR polymorphism in human diabetes. Therefore, we aimed to investigate the effect of natural missense mutations on the expression of human NCB5OR. Protein and mRNA levels of five non-synonymous coding variants were analyzed in transfected HEK293 and HepG2 cells. Although the mRNA levels were only slightly affected by the mutations, the amount of Ncb5or protein was largely reduced upon two Glu to Gly replacements in the third exon (p.E87G, p.E93G). These two mutations remarkably and synergistically shortened the half-life of Ncb5or and their effect could be attenuated by proteasome inhibitors. Our results strongly indicate that p.E87G, p.E93G mutations lead to enhanced proteasomal degradation due to manifest conformational alterations in the b5 domain. These data provide first evidence for natural mutations in NCB5OR gene resulting in decreased protein levels and hence having potential implications in human pathology.