Multi-omic profiling of EPO producing CHO cell panel reveals metabolic adaptation to heterologous protein production

The Chinese hamster ovary (CHO) cell line is the predominant mammalian cell factory for production of therapeutic glycoproteins. In this work, we aimed to study bottlenecks in the secretory pathway associated with the production of human erythropoietin (EPO) in CHO cells. In connection to this, we discovered indications of metabolic adaptation of the amino acid catabolism in favor of heterologous protein production. We established a panel of stably EPO expressing CHO-K1 clones spanning a 25-fold productivity range and characterized the clones in batch and chemostat cultures. For this, we employed a multi-­omic physiological characterization including metabolic footprinting of amino acids, metabolite fingerprinting of glycolytic intermediates, NAD(P)H­­/NAD(P)+ and adenosine nucleotide phosphates. We used qPCR, qRT-PCR, western blots and Affymetrix CHO microarrays to assess EPO gene copy numbers, EPO gene expression, intracellular protein levels and genome-wide differential gene expression analysis of genes functionally related to secretory protein processing, respectively. Finally, we generated a network reconstruction of the amino acid catabolism in CHO cells. The reconstruction was utilized as a platform for interpretation of differential gene expression data in a biological meaningful manner. To identify bottlenecks in the protein secretory pathway, we compared EPO gene copy numbers, EPO gene expression levels, intracellular EPO retention and extracellular EPO levels for a high and low producing clone during chemostat culture. The EPO productivity levels were not reflected in EPO gene load, EPO gene expression or intracellular protein retention, indicating that these processes were not limiting EPO productivity. The global gene expression analysis did not identify significant differentially expressed genes related to secretory protein processing. However, when inspecting the gene expression landscape of the amino acid catabolism, we observed an apparent adaptation in favor of EPO production. That is, we discovered that the gene expression levels of amino acid catabolic genes had adapted to preserve the most abundant amino acids in EPO in the high producing ­clone relative to the low producing clone. Based on these data, we speculate that the amino acid metabolism in CHO cells may undergo adaptation in favor of heterologous protein production during long-term cultivation

Assimilation, Cultural Pluralism and Social Exclusion Among Ethno-Cultural Groups

Abstract: In this paper we use custom tabulations from the 1991 Census for Greater Vancouver to compare the settlement experience of immigrants with ethnic origins in Europe (the 'traditional' stream) and outside Europe (the 'non-traditional' stream). In particular we analyze the extent to which assimilation or cultural pluralism best describe the differential experience of the two groups. Assimilation is measured according to the degree to which either group moves toward the characteristics of the native-born population, while cultural pluralism is assessed from profiles of residential concentration, employment segmentation, mother-tongue retention and ethnic in-marriage. To add a dynamic component, traditional and non-traditional ethnicities are divided into three cohorts according to their length of residence in Canada. We also assess the extent to which assimilation or cultural pluralism is associated with social exclusion, that is, marginalization in terms of economic and educational achievement. Many trends emerge from the complex inter-correlations between these sets of variables. In general we find that assimilation best describes the experience of both groupings, though it is much slower for non-European immigrants and ethnicities, where cultural pluralism survives appreciably beyond the first generation. Cultural pluralism is associated with economic marginality for both groups in their first decade in Canada, though more profoundly for non-European immigrants in terms of personal income. However, labour power is substituted for human capital and household incomes among non-traditional ethnicities exceed those of European-origin groups after a decade of residence. In contrast there is some evidence that for the European-origin native-born, some ethnic separation remains and is associated with economic privilege. In general with length of residence, the relationship between variables becomes more ordered, and education emerges as a structuring effect in shaping economic outcomes. In the early years of immigration, in contrast, education has very little predictive power in terms of economic achievement

A new view of responses to first-time barefoot running.

We examined acute alterations in gait and oxygen cost from shod-to-barefoot running in habitually-shod well-trained runners with no prior experience of running barefoot. Thirteen runners completed six-minute treadmill runs shod and barefoot on separate days at a mean speed of 12.5 km·h-1. Steady-state oxygen cost in the final minute was recorded. Kinematic data were captured from 30-consecutive strides. Mean differences between conditions were estimated with 90% confidence intervals. When barefoot, stride length and ground-contact time decreased while stride rate increased. Leg-and vertical stiffness and ankle-mid-stance dorsi-flexion angle increased when barefoot while horizontal distance between point of contact and the hip decreased. Mean oxygen cost decreased in barefoot compared to shod running (90% CI -11% to -3%) and was related to change in ankle angle and point-of-contact distance, though individual variability was high (-19% to +8%). The results suggest that removal of shoes produces an alteration in running gait and a potentially-practically-beneficial reduction in mean oxygen cost of running in trained-habitually-shod runners new to running barefoot. However, high variability suggests an element of skill in adapting to the novel task and that caution be exercised in assuming the mean response applies to all runners

A New World for Chemical Synthesis?

This perspective seeks to provide an overarching vision of the current state of chemical synthesis methodology using machinery as enabling tools. It highlights current capabilities and limitations in this highly digitally-connected world and suggests areas where new opportunities may arise in the future by going well beyond our present levels of innovation and automation. There is a new need for improved downstream processing tools, advanced reactor design, computational predictive algorithms and integration of robotic systems to maximise the human resource to facilitate a new era in the assembly of our functional materials.The authors gratefully acknowledge financial support from Pfizer (Y.C.), DSTL (O. M.) and the H2020-FET OPEN-2016-2017 Programme of the European commission (D.E.F., S.V.L.;grant agreement number:737266-ONE FLOW

Expression profiling of snoRNAs in normal hematopoiesis and AML

Key Points A subset of snoRNAs is expressed in a developmental- and lineage-specific manner during human hematopoiesis. Neither host gene expression nor alternative splicing accounted for the observed differential expression of snoRNAs in a subset of AML.</jats:p

Stochastic Ion Acceleration by the Ion-cyclotron Instability in a Growing Magnetic Field

Using 1D and 2D particle-in-cell (PIC) simulations of a plasma with a growing magnetic field $\vec{B}$, we show that ions can be stochastically accelerated by the ion-cyclotron (IC) instability. As $\vec{B}$ grows, an ion pressure anisotropy $p_{\perp,i} > p_{||,i}$ arises, due to the adiabatic invariance of the ion magnetic moment ($p_{||,i}$ and $p_{\perp,i}$ are the ion pressures parallel and perpendicular to $\vec{B}$). When initially $\beta_i = 0.5$ ($\beta_i \equiv 8\pi p_i/|\vec{B}|^2$, where $p_i$ is the ion isotropic pressure), the pressure anisotropy is limited mainly by inelastic pitch-angle scattering provided by the IC instability, which in turn produces a non-thermal tail in the ion energy spectrum. After $\vec{B}$ is amplified by a factor $\sim 2.7$, this tail can be approximated as a power-law of index $\sim 3.4$ plus two non-thermal bumps, and accounts for $2-3\%$ of the ions and $\sim 18\%$ of their kinetic energy. On the contrary, when initially $\beta_i =2$, the ion scattering is dominated by the mirror instability and the acceleration is suppressed. This implies that efficient ion acceleration requires that initially $\beta_i \lesssim 1$. Although we focus on cases where $\vec{B}$ is amplified by plasma shear, we check that the acceleration occurs similarly if $\vec{B}$ grows due to plasma compression. Our results are valid in a sub-relativistic regime where the ion thermal energy is $\sim 10\%$ of the ion rest mass energy. This acceleration process can thus be relevant in the inner region of low-luminosity accretion flows around black holes

Organic synthesis: march of the machines.

Organic synthesis is changing; in a world where budgets are constrained and the environmental impacts of practice are scrutinized, it is increasingly recognized that the efficient use of human resource is just as important as material use. New technologies and machines have found use as methods for transforming the way we work, addressing these issues encountered in research laboratories by enabling chemists to adopt a more holistic systems approach in their work. Modern developments in this area promote a multi-disciplinary approach and work is more efficient as a result. This Review focuses on the concepts, procedures and methods that have far-reaching implications in the chemistry world. Technologies have been grouped as topics of opportunity and their recent applications in innovative research laboratories are described.The authors gratefully acknowledge support from UK Engineering and Physical Sciences Research Council (SVL and RMM), Woolf Fisher Trust (DEF) and Pfizer Worldwide Research and Development (CB, RJI).This is the accepted manuscript. The final version is available at http://onlinelibrary.wiley.com/doi/10.1002/anie.201410744/abstract

Machine-Assisted Organic Synthesis.

In this Review we describe how the advent of machines is impacting on organic synthesis programs, with particular emphasis on the practical issues associated with the design of chemical reactors. In the rapidly changing, multivariant environment of the research laboratory, equipment needs to be modular to accommodate high and low temperatures and pressures, enzymes, multiphase systems, slurries, gases, and organometallic compounds. Additional technologies have been developed to facilitate more specialized reaction techniques such as electrochemical and photochemical methods. All of these areas create both opportunities and challenges during adoption as enabling technologies