Comparison of l‐tyrosine containing dipeptides reveals maximum ATP availability for l‐prolyl‐l‐tyrosine in CHO cells

Increasing markets for biopharmaceuticals, including monoclonal antibodies, have triggered a permanent need for bioprocess optimization. Biochemical engineering approaches often include the optimization of basal and feed media to improve productivities of Chinese hamster ovary (CHO) cell cultures. Often, l ‐tyrosine is added as dipeptide to deal with its poor solubility at neutral pH. Showcasing IgG1 production with CHO cells, we investigated the supplementation of three l ‐tyrosine (TYR, Y) containing dipeptides: glycyl‐l ‐tyrosine (GY), l ‐tyrosyl‐l ‐valine (YV), and l ‐prolyl‐l ‐tyrosine (PY). While GY and YV led to almost no phenotypic and metabolic differences compared to reference samples, PY significantly amplified TYR uptake thus maximizing related catabolic activity. Consequently, ATP formation was roughly four times higher upon PY application than in reference samples.     

Evaluation of Protein Profiles From Treated Xenograft Tumor Models Identifies an Antibody Panel for Formalin-fixed and Paraffin-embedded (FFPE) Tissue Analysis by Reverse Phase Protein Arrays (RPPA)

Reverse phase protein arrays (RPPA) are an established tool for measuring the expression and activation status of multiple proteins in parallel using only very small amounts of tissue. Several studies have demonstrated the value of this technique for signaling pathway analysis using proteins extracted from fresh frozen (FF) tissue in line with validated antibodies for this tissue type; however, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation in the clinical setting. Hence, we performed RPPA to measure profiles for a set of 300 protein markers using matched FF and FFPE tissue specimens to identify which markers performed similarly using the RPPA technique in fixed and unfixed tissues. Protein lysates were prepared from matched FF and FFPE tissue specimens of individual tumors taken from three different xenograft models of human cancer. Materials from both untreated mice and mice treated with either anti-HER3 or bispecific anti-IGF-1R/EGFR monoclonal antibodies were analyzed. Correlations between signals from FF and FFPE tissue samples were investigated. Overall, 60 markers were identified that produced comparable profiles between FF and FFPE tissues, demonstrating significant correlation between the two sample types. The top 25 markers also showed significance after correction for multiple testing. The panel of markers covered several clinically relevant tumor signaling pathways and both phosphorylated and nonphosphorylated proteins were represented. Biologically relevant changes in marker expression were noted when RPPA profiles from treated and untreated xenografts were compared. These data demonstrate that, using appropriately selected antibodies, RPPA analysis from FFPE tissue is well feasible and generates biologically meaningful information. The identified panel of markers that generate similar profiles in matched fixed and unfixed tissue samples may be clinically useful for pharmacodynamic studies of drug effect using FFPE tissues.Many human diseases are characterized by abnormalities in complex signaling pathways (1). The expression and activation status of proteins from these deregulated pathways has traditionally been analyzed using single marker techniques such as immunohistochemistry and Western blotting. Although these techniques have provided valuable information on the molecular abnormalities underlying human disease, they are labor intensive, have a low throughput, and often require high sample volume. Furthermore, techniques such as Western blotting are not applicable in the routine clinical setting. Miniaturized parallel immunoassay techniques have been developed in recent years and have played a pivotal role in biomarker discovery (2). Antibody arrays enable multiple potential disease markers to be investigated in a single sample in parallel (3). Beyond this, Reverse Phase Protein Arrays (RPPA)¹ are sensitive high throughput tools that can quantify protein expression levels and activation status (posttranslational modifications such as phosphorylation) in multiple experimental samples simultaneously. The technique requires only minute amounts of samples, printed as lysate arrays onto slides, and hundreds of markers of interest can be investigated, array by array, in a miniaturized dot blot manner. Numerous reports have demonstrated that RPPA can be applied to various sources of cells and tissues to analyze protein profiles, signaling pathway networks, and for the identification of biomarkers (4–13). A recently published workshop report reviews the full potential and advances of RPPA for use in clinical, translational, and basic research (11).In oncology, the parallel profiling of multiple protein markers is particularly desirable to study tumor initiation and progression, to classify tumor disease states on the molecular level, and to discover and monitor biomarkers that can predict therapeutic response or tumor recurrence (14–16). The study of signaling response and analysis of pharmacodynamic (PD) markers upon treatment using in vitro and in vivo test systems (e.g. cell line or patient derived xenograft tumor models) is an established component of preclinical and early clinical drug development. These techniques can provide evidence of target pathway modulation for new therapeutic lead candidate compounds and provide valuable information on the drug mode of action (17), especially in the translational phase. Multiplex analyses of PD biomarkers by RPPA have been performed in vitro using cancer cell lines (18, 19) as well as in patient-derived tumor tissue and blood samples (20, 21) to assess response to treatment and target inhibition. A combination of RPPA signaling pathway mapping and functional PET imaging has recently been successfully evaluated in xenograft models as an early response PD marker for anti-cancer drug efficacy (13).Translating miniaturized multiple protein analysis platforms-such as RPPA - from preclinical to clinical applicability is highly desirable; however, issues such as the limited amount of available clinical samples and tumor heterogeneity must first be addressed. Furthermore, most studies of RPPA in tumor tissue to date have been conducted using proteins extracted from fresh-frozen (FF) tissue specimens; whereas, formalin fixation and paraffin embedding (FFPE) is the standard method for tissue preservation used in clinical pathology laboratories. FFPE yields excellent tissue architecture for histological assessment and enables analysis of individual proteins in situ by techniques such as immunohistochemistry. However, formalin fixation leads to extensive protein–protein and protein–nucleic acid cross-linking (22), which can hamper protein extraction and reduce both the overall yield of extracted protein and the profile of proteins detectable by proteomic techniques (23, 24). Furthermore, formalin-induced cross-linking induces conformational changes in protein structure that can alter the immunoreactivity of some proteins in situ by hiding or altering peptide epitopes (25, 26). Such artifacts are absent from snap-frozen tissue; therefore, protein profiles obtained from FF tissue are likely to reflect the in vivo biology of the tumor more closely. However, FF tumor tissue is not widely available because it is costly to collect and maintain in the clinical setting. FFPE tissue samples are routinely archived by nearly every hospital and offer a unique opportunity to study thousands of samples retrospectively with extensive clinical records and follow-up information.Several groups have now established protocols for retrieving cross-linked proteins from fixed tissues (27–33). These methods are mainly based on the use of concentrated ionic detergents and high temperature protocols closely related to the antigen retrieval methods developed for immunohistochemistry. These studies show that obtaining nondegraded, full-length proteins from FFPE tissues for multiplex analyses is feasible (27–33). More recently, protein extraction techniques optimized for fixed samples have been used to successfully conduct RPPA using FFPE tissue biopsies from different cancer types (34–40). Guo et al. systematically investigated several protein extraction methods and demonstrated that RPPA of FFPE materials is feasible, reproducible and can generate biologically relevant protein profiles (41). Other studies have confirmed the validity of this approach and shown that data generated from RPPA analyses of FFPE tissue demonstrate good concordance with traditional immunohistochemistry markers such as HER2 protein in breast cancer (34, 40). However, to date, analyses have been performed only for a limited set of protein markers.To evaluate whether analysis of a broader panel of protein markers is feasible and generates meaningful data from FFPE tumor tissue sections, we conducted RPPA on matched samples of FF and FFPE tissues using a set of 300 markers, the largest panel reported to date. Our aim was to identify markers that performed similarly when comparing the protein profiles measured in protein extracts from matched FF and FFPE tissue, using RPPA assays established for use in frozen tissues. Correlating selected markers and assays in such a way should qualify RPPA for further use with FFPE tissues of clinical relevance, e.g. in PD marker studies. In this paper, we have specifically focused on the technical issues relevant for using the RPPA platform in a clinical setting, and did not address the biology of the test systems used in detail. However, the models used have been pre-characterized to identify key signaling parameters in context of targeted drug treatment (42). We conducted a systematic comparison of RPPA protein profiles in matched FF and FFPE tumor tissues resected from three different xenograft models of human cancer, each treated with targeted therapeutic antibodies that have previously been shown to achieve tumor growth inhibition. Furthermore, we investigated the effect of targeted drug treatment on protein expression and activation status, and the concordance of matched FF and FFPE tissue RPPA profiles. Finally, with one of the applied tumor models, we compared a set of protein profiles measured with two different multiple assay platforms - the RPPA and the Luminex Bio-Plex system, and discuss their relevance with respect to the analysis of FFPE tissue.     

Doing Philosophy Effectively: Student Learning in Classroom Teaching

An important aim of teaching philosophy in Dutch secondary schools is to learn about philosophy (i.e., the great philosophers) by doing philosophy. We examined doing philosophy and focused specifically on the relationship between student learning activities and teacher behavior; in doing so, a qualitative cross-case analysis of eight philosophy lessons was performed. The effectiveness of doing philosophy was operationalized into five learning activities comprising rationalizing, analyzing, testing, producing criticism, and reflecting, and scored by means of qualitative graphical time registration. Using CA we find a quantitative one-dimensional scale for the lessons that contrasts lessons that are more and less effective in terms of learning and teaching. A relationship was found between teaching by teachers and doing philosophy by students. In particular we found students to produce a higher level of doing philosophy with teachers who chose to organize a philosophical discussion with shared guidance by the teacher together with the students.     

Macroecological patterns of archaeal ammonia oxidizers in the Atlantic Ocean

Macroecological patterns are found in animals and plants, but also in micro‐organisms. Macroecological and biogeographic distribution patterns in marine Archaea, however, have not been studied yet. Ammonia‐oxidizing Archaea (AOA) show a bipolar distribution (i.e. similar communities in the northernmost and the southernmost locations, separated by distinct communities in the tropical and gyral regions) throughout the Atlantic, detectable from epipelagic to upper bathypelagic layers (<2000 m depth). This tentatively suggests an influence of the epipelagic conditions of organic matter production on bathypelagic AOA communities. The AOA communities below 2000 m depth showed a less pronounced biogeographic distribution pattern than the upper 2000 m water column. Overall, AOA in the surface and deep Atlantic waters exhibit distance–decay relationships and follow the Rapoport rule in a similar way as bacterial communities and macroorganisms. This indicates a major role of environmental conditions in shaping the community composition and assembly (species sorting) and no, or only weak limits for dispersal in the oceanic thaumarchaeal communities. However, there is indication of a different strength of these relationships between AOA and Bacteria, linked to the intrinsic differences between these two domains.     

The Net Landscape Carbon Balance—Integrating terrestrial and aquatic carbon fluxes in a managed boreal forest landscape in Sweden

The boreal biome exchanges large amounts of carbon (C) and greenhouse gases (GHGs) with the atmosphere and thus significantly affects the global climate. A managed boreal landscape consists of various sinks and sources of carbon dioxide (CO₂), methane (CH₄), and dissolved organic and inorganic carbon (DOC and DIC) across forests, mires, lakes, and streams. Due to the spatial heterogeneity, large uncertainties exist regarding the net landscape carbon balance (NLCB). In this study, we compiled terrestrial and aquatic fluxes of CO₂, CH₄, DOC, DIC, and harvested C obtained from tall‐tower eddy covariance measurements, stream monitoring, and remote sensing of biomass stocks for an entire boreal catchment (~68 km²) in Sweden to estimate the NLCB across the land–water–atmosphere continuum. Our results showed that this managed boreal forest landscape was a net C sink (NLCB = 39 g C m^?2 year^?1) with the landscape–atmosphere CO₂ exchange being the dominant component, followed by the C export via harvest and streams. Accounting for the global warming potential of CH₄, the landscape was a GHG sink of 237 g CO₂‐eq m^?2 year^?1, thus providing a climate‐cooling effect. The CH₄ flux contribution to the annual GHG budget increased from 0.6% during spring to 3.2% during winter. The aquatic C loss was most significant during spring contributing 8% to the annual NLCB. We further found that abiotic controls (e.g., air temperature and incoming radiation) regulated the temporal variability of the NLCB whereas land cover types (e.g., mire vs. forest) and management practices (e.g., clear‐cutting) determined their spatial variability. Our study advocates the need for integrating terrestrial and aquatic fluxes at the landscape scale based on tall‐tower eddy covariance measurements combined with biomass stock and stream monitoring to develop a holistic understanding of the NLCB of managed boreal forest landscapes and to better evaluate their potential for mitigating climate change.     

S-adenosylmethionine and methylthioadenosine boost cellular productivities of antibody forming Chinese hamster ovary cells

The improvement of cell specific productivities for the formation of therapeutic proteins is an important step towards intensified production processes. Among others, the induction of the desired production phenotype via proper media additives is a feasible solution provided that said compounds adequately trigger metabolic and regulatory programs inside the cells. In this study, S-(5′-adenosyl)- l -methionine (SAM) and 5′-deoxy-5′-(methylthio)adenosine (MTA) were found to stimulate cell specific productivities up to approx. 50% while keeping viable cell densities transiently high and partially arresting the cell cycle in an anti-IL-8-producing CHO-DP12 cell line. Noteworthy, MTA turned out to be the chemical degradation product of the methyl group donor SAM and is consumed by the cells.     

Association between sexually selected traits and allelic distance in two unlinked MHC II loci in white-tailed deer (Odocoileus virginianus)

Evolutionary Ecology - Body size and secondary sexual characteristics are drivers of male reproductive success among polygynous species. A gene complex found to be associated with morphology in...     

Synthesis and characterization of several carbamoyl- and methylcarbamoyl-substituted EMPO derivatives

The spin trapping behavior of four novel carbamoyl-substituted EMPO derivatives, namely 5-carbamoyl-3,5-dimethyl-pyrroline N-oxide (CADMPO), 3,5-dimethyl-5-methylcarbamoyl-pyrroline N-oxide (DMMCAPO), 5-carbamoyl-3-ethyl-5-methyl-pyrroline N-oxide (CAEMPO), and 3-ethyl-5-methyl-5-methylcarbamoyl-pyrroline N-oxide (EMMCAPO), towards different oxygen- and carbon-centered radicals is described, the half lives of the respective superoxide adducts ranging from about 10 to 20 min. The most characteristic adducts were, however, formed from methyl, hydroxymethyl, hydroxyethyl, and carbon dioxide anion radicals.     

Response of Net Ecosystem Productivity of Three Boreal Forest Stands to Drought

In 2001–03, continuous eddy covariance measurements of carbon dioxide (CO₂) flux were made above mature boreal aspen, black spruce, and jack pine forests in Saskatchewan, Canada, prior to and during a 3−year drought. During the 1st drought year, ecosystem respiration (R) was reduced at the aspen site due to the drying of surface soil layers. Gross ecosystem photosynthesis (GEP) increased as a result of a warm spring and a slow decrease of deep soil moisture. These conditions resulted in the highest annual net ecosystem productivity (NEP) in the 9 years of flux measurements at this site. During 2002 and 2003, a reduction of 6% and 34% in NEP, respectively, compared to 2000 was observed as the result of reductions in both R and GEP, indicating a conservative response to the drought. Although the drought affected most of western Canada, there was considerable spatial variability in summer rainfall over the 100−km extent of the study area; summer rainfalls in 2001 and 2002 at the two conifer sites minimized the impact of the drought. In 2003, however, precipitation was similarly low at all three sites. Due to low topographic position and consequent poor drainage at the black spruce site and the coarse soil with low water-holding capacity at the jack pine site almost no reduction in R, GEP, and NEP was observed at these two sites. This study shows that the impact of drought on carbon sequestration by boreal forest ecosystems strongly depends on rainfall distribution, soil characteristics, topography, and the presence of vegetation that is well adapted to these conditions.     

Response of Net Ecosystem Productivity of Three Boreal Forest Stands to Drought

In 2000–03, continuous eddy covariance measurements of carbon dioxide (CO₂) flux were made above mature boreal aspen, black spruce, and jack pine forests in Saskatchewan, Canada, prior to and during a 3-year drought. During the 1st drought year, ecosystem respiration (R) was reduced at the aspen site due to the drying of surface soil layers. Gross ecosystem photosynthesis (GEP) increased as a result of a warm spring and a slow decrease of deep soil moisture. These conditions resulted in the highest annual net ecosystem productivity (NEP) in the 9 years of flux measurements at this site. During 2002 and 2003, a reduction of 6% and 34% in NEP, respectively, compared to 2000 was observed as the result of reductions in both R and GEP, indicating a conservative response to the drought. Although the drought affected most of western Canada, there was considerable spatial variability in summer rainfall over the 100-km extent of the study area; summer rainfalls in 2001 and 2002 at the two conifer sites minimized the impact of the drought. In 2003, however, precipitation was similarly low at all three sites. Due to low topographic position and consequent poor drainage at the black spruce site and the coarse soil with low water-holding capacity at the jack pine site almost no reduction in R, GEP, and NEP was observed at these two sites. This study shows that the impact of drought on carbon sequestration by boreal forest ecosystems strongly depends on rainfall distribution, soil characteristics, topography, and the presence of vegetation that is well adapted to these conditions. The online version of the original article can be found under doi: