SUMMARY Chinese hamster ovary (CHO) cells dominate biotherapeutic protein production and are widely used in mammalian cell line engineering research. To elucidate metabolic bottlenecks in protein production and to guide cell engineering and bioprocess optimization, we reconstructed the metabolic pathways in CHO and associated them with >1,700 genes in the Cricetulus griseus genome. The genome-scale metabolic model based on this reconstruction, iCHO1766, and cell line-specific models for CHO-K1, CHO-S, and CHO-DG44 cells, provide the biochemical basis of growth and recombinant protein production. The models accurately predict growth phenotypes and known auxotrophies in CHO cells. With the models, we quantify the protein synthesis capacity of CHO cells and demonstrate that common bioprocess treatments, such as histone deacetylase inhibitors, inefficiently increase product yield. However, our simulations show the metabolic resources in CHO are >3 times more efficiently utilized for growth or recombinant protein synthesis following targeted efforts to engineer the CHO secretory pathway. This model will further accelerate CHO cell engineering and help optimize bioprocesses.

Four members (SOCS-1, SOCS-2, SOCS-3, and CIS) of a family of cytokine-inducible, negative regulators of cytokine receptor signaling have recently been identified. To address whether any of these genes are induced in response to growth hormone (GH), serum-starved 3T3-F442A fibroblasts were incubated with GH for various time points, and the expression of the SOCS gene family was analyzed by Northern blotting. GH stimulated the rapid, transient induction of SOCS-3 mRNA, peaking 30 min after the initiation of GH exposure and declining to basal levels by 2 h. Expression of the other SOCS genes (SOCS-1, SOCS-2, CIS) was also up-regulated by GH, although to a lesser extent than SOCS-3 and with differing kinetics. SOCS-3 expression was also strongly induced in 3T3-F442A cells treated with leukemia-inhibitory factor (LIF), with weaker induction of SOCS-1 and CIS being observed. The preferential induction of SOCS-3 mRNA was also observed in hepatic RNA isolated from the livers of mice that had received a single supraphysiological dose of GH intraperitoneally. Co-transfection studies revealed that constitutive expression of SOCS-1 and SOCS-3, but not SOCS-2 or CIS, blocked GH-induced transactivation of the GH-responsive serine protease inhibitor 2.1 gene promoter.The elucidation of the pivotal role played by the tyrosine kinase Jak2 in initiating signal transduction from the GH 1 receptor has led to the identification of a number of intracellular pathways that mediate the cellular response to GH (1). However, the mechanism(s) by which signaling from GH receptor-activated Jak2 is attenuated is unclear. Ligand-induced tyrosine phosphorylation/activation of Jak2 by the erythropoietin (EPO) receptor, a member of the cytokine receptor superfamily that includes the GH receptor, is followed by the binding of the protein-tyrosine phosphatase SHP-1 to the cytoplasmic domain of the receptor (2). The recruitment of SHP-1 is accompanied by the dephosphorylation/inactivation of Jak2 and subsequent termination of EPO-induced cellular proliferation.A similar role for SHP-1 in mediating the down-regulation of Jak2 following stimulation of cells with GH has been proposed (3), although whether SHP-1 can directly associate with the GH receptor remains to be established. Recently, a novel family of cytokine-inducible genes has been identified that appear to function as negative regulators of the JAK signaling pathway (4 -7). Constitutive expression of one member, SOCS-1 (also referred to as SSI-1 and JAB) in the murine myeloid leukemia M1 cell line blocked growth factor-induced differentiation and apoptosis and inhibited interleukin-6 (IL-6)-mediated tyrosine phosphorylation of the cell-surface receptor component, gp130, and the transcription factor, Stat3 (5, 6). SOCS-1 can interact with all four members of the JAK family of tyrosine kinases (6, 7), suppressing kinase activity and the subsequent tyrosine phosphorylation/activation of STAT factors (7). As a diverse array of cytokines appears to be able to induce expression of one or more mem...

This paper presents results from lean CO/H 2 /O 2 /NO x oxidation experiments conducted at 20-100 bar and 600-900 K. The experiments were carried out in a new high-pressure laminar flow reactor designed to conduct well-defined experimental investigations of homogeneous gas phase chemistry at pressures and temperatures up to 100 bar and 925 K. at 780-1100 K and 1-10 bar. Moreover, introduction of the reaction CO + H 2 O 2 → HOCO + OH into the model yields an improved prediction, but no final resolution, to the recently debated syngas ignition delay problem compared to previous kinetic models.

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Straw is used as fuel in relatively small-scale combined heat and power producing (CHP) grate boilers in Denmark. The large content of potassium and chlorine in straw greatly increases the deposit formation and corrosion of the superheater coils, compared to boilers firing coal. In this study, mature superheater deposit samples were extracted from two straw-fired boilers, Masnedø and Ensted, with fuel inputs of 33 MWth and 100 MWth, respectively. SEM (scanning electron microscopy) images and EDX (energy dispersive X-ray) analyses were performed on the deposit samples. Different strategies are adopted to minimize deposit problems at the two boilers. At Masnedø the final superheater steam temperature is 520 °C, no soot blowing of the superheaters is applied and a relatively large superheater area is used. At Ensted, an external wood-fired superheater is used in order to obtain a final steam temperature of 542 °C, while the steam exit temperature of the straw-fired boiler is 470 °C. The mature Masnedø deposit had a thickness of 2 to 15 centimeters and consisted of three distinct main layers. The thick intermediate layer was depleted in chlorine but rich in Si, K, and Ca. This Masnedø intermediate layer was probably generated by in-situ reaction between KCl and Si-rich ash particles, which leads to release of chlorine-containing gases. The innermost layer contained many sublayers of mainly iron oxide, KCl, and K2SO4. The Ensted deposit had a maximum thickness of a few centimeters. The intermediate Ensted layer consisted of melted KCl with inclusions of Ca- and Si-rich particles, and the innermost layer was an iron oxide next to a potassium sulfate layer. Compared to deposits formed on a probe during short-time experiments, the mature superheater deposits contained larger dense inner sublayers of pure KCl and K2SO4. The present study indicates that the innermost layer of the superheater deposits expands by condensation of KCl, even when the deposit has a thickness of several centimeters.

In this study we have investigated the role of suppressor of cytokine signaling (SOCS) proteins in GH receptor-mediated signaling. GH-induced transcription was inhibited by SOCS-1 and SOCS-3, while SOCS-2 and cytokine inducible SH2-containing protein (CIS) had no effect By using chimeric SOCS proteins it was found that the ability of SOCS proteins to inhibit GH-mediated transcription was located in the amino-terminal 40-80 amino acids. In SOCS-3, 46 amino acids C-terminal to the SH2 domain were required for the inhibitory activity, while a truncated SOCS-1 having only 2 amino acids C-terminal to the SH2 domain was able to inhibit GH-mediated transcription. Both SOCS-1 and SOCS-3 were able to inhibit GH-induced STAT5 (signal transducer and activator of transcription) activation. SOCS-1 inhibited the tyrosine kinase activity of Janus kinase 2 (JAK2) directly, while SOCS-3 only inhibited JAK2 when stimulated by the GH receptor. All four SOCS proteins were able to bind to a tyrosine-phosphorylated glutathione-S-transferase-GH receptor fusion protein, and SOCS-3 required the same 46 C-terminal amino acids for GH receptor binding as it did for inhibition of GH-mediated transcription and STAT5 activation. These data suggest that SOCS-1 and -3 can suppress GH-induced transcriptional activity, presumably by inhibiting the kinase activity of JAK2 either directly in the case of SOCS-1 or via binding to the tyrosine-phosphorylated GH receptor in the case of SOCS-3.

Glycosylation is a critical quality attribute of most recombinant biotherapeutics. Consequently, drug development requires careful control of glycoforms to meet bioactivity and biosafety requirements. However, glycoengineering can be extraordinarily difficult given the complex reaction networks underlying glycosylation and the vast number of different glycans that can be synthesized in a host cell. Computational modeling offers an intriguing option to rationally guide glycoengineering, but the high parametric demands of current modeling approaches pose challenges to their application. Here we present a novel low-parameter approach to describe glycosylation using flux-balance and Markov chain modeling. The model recapitulates the biological complexity of glycosylation, but does not require user-provided kinetic information. We use this method to predict and experimentally validate glycoprofiles on EPO, IgG as well as the endogenous secretome following glycosyltransferase knock-out in different Chinese hamster ovary (CHO) cell lines. Our approach offers a flexible and user-friendly platform that can serve as a basis for powerful computational engineering efforts in mammalian cell factories for biopharmaceutical production.

Signal transducer and activator of transcription 5 (STAT5) activation plays a central role in GH- and prolactin-mediated signal transduction in the pancreatic beta-cells. In previous experiments we demonstrated that STAT5 activation is necessary for human (h)GH-stimulated proliferation of INS-1 cells and hGH-induced increase of mRNA-levels of the cell cycle regulator cyclin D2. In this study we have further characterized the role of STAT5 in the regulation of cyclin D expression and beta-cell proliferation by hGH. Cyclin D2 mRNA and protein levels (but not cyclin D1 and D3) were induced in a time-dependent manner by hGH in INS-1 cells. Inhibition of protein synthesis by coincubation with cycloheximide did not affect the hGH-induced increase of cyclin D2 mRNA levels at 4 h. Expression of a dominant negative STAT5 mutant, STAT5aDelta749, partially inhibited cyclin D2 protein levels. INS-1 cells transiently transfected with a cyclin D2 promoter-reporter construct revealed a 3- to 5-fold increase of transcriptional activity in response to hGH stimulation. Furthermore, coexpression of a constitutive active STAT5 mutant (either CA-STAT5a or CA-STAT5b) was sufficient to drive transactivation of the promoter. CA-STAT5b was stably expressed in INS-1 cells under the control of a doxycycline-inducible promoter. Gel retardation experiments using a probe representing a putative STAT5 binding site in the cyclin D2 promoter revealed binding of the doxycycline-induced CA-STAT5b. Furthermore, induction of CA-STAT5b stimulated transcriptional activation of the cyclin D2 promoter and induced hGH-independent proliferation in these cells. In primary beta-cells, adenovirus-mediated expression of CA-STAT5b profoundly stimulated DNA-synthesis (5.3-fold over control) in the absence of hGH. Our studies indicate that STAT5 activation is sufficient to drive proliferation of the beta-cells and that cyclin D2 may be a critical target gene for STAT5 in this process.