Analysis of DNA DSB repair and production stability in CHO cells

Poster Number 13 ANALYSIS OF DNA DSB REPAIR AND PRODUCTION STABILITY IN CHO CELLS Xiaolin Zhang, Department of Chemical and Biomolecular Engineering, University of Delaware Delaware Biotechnology Institute, University of Delaware [email protected] Kelvin H. Lee, Department of Chemical and Biomolecular Engineering, University of Delaware Delaware Biotechnology Institute, University of Delaware Key Words: CHO cell, DSB repair, heterologous expression, production instability. Productivity of recombinant proteins in CHO cell lines often decreases over long-term cultivation. This production instability limits the use of CHO-based platforms and can negatively impact the capability of a manufacturing process to meet market demands. A method to prevent the production loss during long-term cultivation is highly desirable. Genome instability can reduce transgene copy number and is reported as a major cause for production instability. We hypothesize that the DNA double-strand break (DSB) repair system in CHO is deficient and associated with both genome and production instabilities. Our results indicated that CHO cells had a lower DSB repair rate compared to the bEnd.3 mouse endothelial cell line, which is consistent with our hypothesis. The ability to improve DSB repair in CHO may provide a strategy to prevent production instability. Therefore, we tested heterologous expression of eight DSB repair-related genes, and found that four genes could significantly improve DSB repair in CHO cells. To further assess the impact of improved DSB repair on protein production, each of the four heterologous genes was stably expressed in a secreted alkaline phosphatase (SEAP) producing cell line, and SEAP production in single clones was evaluated over three months in the absence of methotrexate (MTX). Our results showed that productivity correlated strongly with the SEAP copy number, and two heterologous genes could substantially improve the production retention during long-term cultivation

Investigations of urethral sphincter activity in mice with bladder hyperalgesia before and after drug administration of gabapentin.

PurposeThis study investigated the effect of gabapentin on lower urinary tract dysfunction focusing on urethral activities and cystitis-induced hyperalgesia in a mouse model of painful bladder syndrome/interstitial cystitis (PBS/IC). The electromyography (EMG) of external urethral sphincter (EUS) was difficult to obtain, but contained useful information to examine the drug effect in mice.MethodsFemale C57BL/6J mice were intraperitoneally (ip) administration with either saline or 200 mg/kg of cyclophosphamide (CYP) 48 h before experimental evaluation. Cystitis mice were treated with administration of gabapentin (25 or 50 mg/kg, ip). Cystometry and EUS EMG were obtained and analyzed during continuous bladder infusion. The visceral pain-related visceromotor reflex (VMR) was recorded in response to isotonic bladder distension.ResultsCystitis mice showed shorter inter-contraction intervals and increased occurrence of non-voiding contractions during bladder infusion, with increased VMR during isotonic bladder distension, indicating cystitis-induced bladder hyperalgesia. Gabapentin (50 mg/kg) suppressed effects of CYP on cystometry, but not on EUS EMG activity, during bladder infusion. The effect on urodynamic recordings lasted 4 h. VMR was significantly reduced by gabapentin.ConclusionsThe present study showed that CYP-induced cystitis in mice is a model of visceral hyperalgesia affecting detrusor contractions, not urethral activations. The technique of using EUS EMG to evaluate the drug effects on urethral activities is novel and useful for future investigations. Gabapentin can be as a potential treatment for detrusor overactivity and PBS/IC

Five-S-isotope evidence of two distinct mass-independent sulfur isotope effects and implications for the modern and Archean atmospheres.

The signature of mass-independent fractionation of quadruple sulfur stable isotopes (S-MIF) in Archean rocks, ice cores, and Martian meteorites provides a unique probe of the oxygen and sulfur cycles in the terrestrial and Martian paleoatmospheres. Its mechanistic origin, however, contains some uncertainties. Even for the modern atmosphere, the primary mechanism responsible for the S-MIF observed in nearly all tropospheric sulfates has not been identified. Here we present high-sensitivity measurements of a fifth sulfur isotope, stratospherically produced radiosulfur, along with all four stable sulfur isotopes in the same sulfate aerosols and a suite of chemical species to define sources and mechanisms on a field observational basis. The five-sulfur-isotope and multiple chemical species analysis approach provides strong evidence that S-MIF signatures in tropospheric sulfates are concomitantly affected by two distinct processes: an altitude-dependent positive 33S anomaly, likely linked to stratospheric SO2 photolysis, and a negative 36S anomaly mainly associated with combustion. Our quadruple stable sulfur isotopic measurements in varying coal samples (formed in the Carboniferous, Permian, and Triassic periods) and in SO2 emitted from combustion display normal 33S and 36S, indicating that the observed negative 36S anomalies originate from a previously unknown S-MIF mechanism during combustion (likely recombination reactions) instead of coal itself. The basic chemical physics of S-MIF in both photolytic and thermal reactions and their interplay, which were not explored together in the past, may be another ingredient for providing deeper understanding of the evolution of Earth's atmosphere and life's origin

Re-colonizing spaces of memorializing: the case of the Chattri Indian Memorial, UK

This article inspects the ways that spaces of war memorialization are organized and reorganized through official and unofficial meaning-making activities. It aims to contribute to the discussion of the ‘value’ of memorializing by examining a multifaceted space of remembrance and commemoration: the Chattri Indian Memorial built near Brighton, UK. The article brings postcolonial perspectives to explore how memorializing has been organized here, focusing on the activities of once-colonized people and the affective, embodied aspects of organizing practices. Built in 1921 to honour Indian soldiers who fought in WWI, the Chattri evolved from a colonial instrument to symbol and space for ethnic-Indian group activities. The study employed historical, visual and ethnographic methods to study the tangible monument and the changing nature of the memorializing activities carried out around the monument. Memorializing is conceptualized within three inter-related processes: colonizing, de-colonizing and re-colonizing to examine how forms and practices of memorialization constitute a values-laden organizing system

Very strong intrinsic supercurrent carrying ability and vortex avalanches in (Ba,K)Fe2As2 superconducting single crystals

We report that single crystals of (Ba,K)Fe2As2 with Tc = 32 K have a pinning potential, U0, as high as 10^4 K, with U0 showing very little field depend-ence. In addition, the (Ba,K)Fe2As2 single crystals become isotropic at low temperatures and high magnetic fields, resulting in a very rigid vortex lattice, even in fields very close to Hc2. The rigid vortices in the two dimensional (Ba,K)Fe2As2 distinguish this compound from 2D high Tc cuprate superconductors with 2D vortices, and make it being capable of cearrying very high critical current.Flux jumping due to high Jc was also observed in large samples at low temperatures.Comment: 4 pages, 7 figures. submitte

Constraint-Based Model of Shewanella Oneidensis MR-1 Metabolism: A Tool for Data Analysis and Hypothesis Generation

Shewanellae are gram-negative facultatively anaerobic metal-reducing bacteria commonly found in chemically (i.e., redox) stratified environments. Occupying such niches requires the ability to rapidly acclimate to changes in electron donor/acceptor type and availability; hence, the ability to compete and thrive in such environments must ultimately be reflected in the organization and utilization of electron transfer networks, as well as central and peripheral carbon metabolism. To understand how Shewanella oneidensis MR-1 utilizes its resources, the metabolic network was reconstructed. The resulting network consists of 774 reactions, 783 genes, and 634 unique metabolites and contains biosynthesis pathways for all cell constituents. Using constraint-based modeling, we investigated aerobic growth of S. oneidensis MR-1 on numerous carbon sources. To achieve this, we (i) used experimental data to formulate a biomass equation and estimate cellular ATP requirements, (ii) developed an approach to identify cycles (such as futile cycles and circulations), (iii) classified how reaction usage affects cellular growth, (iv) predicted cellular biomass yields on different carbon sources and compared model predictions to experimental measurements, and (v) used experimental results to refine metabolic fluxes for growth on lactate. The results revealed that aerobic lactate-grown cells of S. oneidensis MR-1 used less efficient enzymes to couple electron transport to proton motive force generation, and possibly operated at least one futile cycle involving malic enzymes. Several examples are provided whereby model predictions were validated by experimental data, in particular the role of serine hydroxymethyltransferase and glycine cleavage system in the metabolism of one-carbon units, and growth on different sources of carbon and energy. This work illustrates how integration of computational and experimental efforts facilitates the understanding of microbial metabolism at a systems level

A significant improvement in both low- and high-field performance of MgB2 superconductors through graphene oxide doping

The effects of graphene oxide (GO) doping on the superconducting properties of MgB2 were studied using bulk samples made by the diffusion method. Homogeneous dispersions of GO in tetrahydrofuran were obtained through a novel synthesis method, which is then chemically doped with MgB2. It was found that GO doping significantly improves the critical current density, under both low and high magnetic fields, which distinguishes GO from all the other elements doped into MgB2 so far. 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved

Transport critical current density in Fe-sheathed nano-SiC doped MgB/sub 2/ wires

The nano-SiC doped MgB/sub 2//Fe wires were fabricated using a powder-in-tube method and an in-situ reaction process. The depression of T/sub c/ with increasing SiC doping level remained rather small due to the counterbalanced effect of Si and C co-doping. The high level SiC co-doping allowed creation of the intra-grain defects and nano-inclusions, which act as effective pinning centers, resulting in a substantial enhancement in the J/sub c/(H) performance. The transport J/sub c/ for all the wires is comparable to the magnetic J/sub c/ at higher fields despite the low density of the samples and percolative nature of current. The transport I/sub c/ for the 10wt% SiC doped MgB/sub 2//Fe reached 660A at 5K and 4.5T (J/sub c/=133000A/cm/sup 2/) and 540A at 20K and 2T (J/sub c/=108000A/cm/sup 2/). The transport J/sub c/ for the 10wt% SiC doped MgB/sub 2/ wire is more than an order of magnitude higher than for the state-the-art Fe-sheathed MgB/sub 2/ wire reported to date at 5K and 10T and 20K and 5T respectively. There is a plenty of room for further improvement in J/sub c/ as the density of the current samples is only 50%