Transgene copy number comparison in recombinant mammalian cell lines: critical reflection of quantitative real-time PCR evaluation

Nucleic acid quantification is a relevant issue for the characterization of mammalian recombinant cell lines and also for the registration of producer clones. Quantitative real-time PCR is a powerful tool to investigate nucleic acid levels but numerous different quantification strategies exist, which sometimes lead to misinterpretation of obtained qPCR data. In contrast to absolute quantification using amplicon- or plasmid standard curves, relative quantification strategies relate the gene of interest to an endogenous reference gene. The relative quantification methods also consider the amplification efficiency for the calculation of the gene copy number and thus more accurate results compared to absolute quantification methods are generated. In this study two recombinant Chinese hamster ovary cell lines were analysed for their transgene copy number using different relative quantification strategies. The individual calculation methods resulted in differences of relative gene copy numbers because efficiency calculations have strong impact on gene copy numbers. However, in context of comparing transgene copy numbers of two individual clones the influence of the calculation method is marginal. Therefore especially for the comparison of two cell lines with the identical transgene any of the relative qPCR methods was proven as powerful tool.     

Immediate and Heterogeneous Response of the LiaFSR Two-Component System of Bacillus subtilis to the Peptide Antibiotic Bacitracin

Background

Two-component signal transduction systems are one means of bacteria to respond to external stimuli. The LiaFSR two-component system of Bacillus subtilis consists of a regular two-component system LiaRS comprising the core Histidine Kinase (HK) LiaS and the Response Regulator (RR) LiaR and additionally the accessory protein LiaF, which acts as a negative regulator of LiaRS-dependent signal transduction. The complete LiaFSR system was shown to respond to various peptide antibiotics interfering with cell wall biosynthesis, including bacitracin.

Methodology and Principal Findings

Here we study the response of the LiaFSR system to various concentrations of the peptide antibiotic bacitracin. Using quantitative fluorescence microscopy, we performed a whole population study analyzed on the single cell level. We investigated switching from the non-induced ‘OFF’ state into the bacitracin-induced ‘ON’ state by monitoring gene expression of a fluorescent reporter from the RR-regulated liaI promoter. We found that switching into the ‘ON’ state occurred within less than 20 min in a well-defined switching window, independent of the bacitracin concentration. The switching rate and the basal expression rate decreased at low bacitracin concentrations, establishing clear heterogeneity 60 min after bacitracin induction. Finally, we performed time-lapse microscopy of single cells confirming the quantitative response as obtained in the whole population analysis for high bacitracin concentrations.

Conclusion

The LiaFSR system exhibits an immediate, heterogeneous and graded response to the inducer bacitracin in the exponential growth phase.     

Ryanodine receptor signaling is required for anti-CD3-induced T cell proliferation, interleukin-2 synthesis, and interleukin-2 receptor signaling

Ryanodine receptors (RyR) are involved in regulating intracellular Ca(++) mobilization in T lymphocytes. However, the importance of RyR signaling during T cell activation has not yet been determined. In this study, we have used the RyR-selective antagonists, ruthenium red and dantrolene, to determine the effect of RyR blockade on T cell receptor-mediated activation events and cytokine-dependent T cell proliferation. Both ruthenium red and dantrolene inhibited DNA synthesis and cell division, as well as the synthesis of interleukin (IL)-2 by T lymphocytes responding to mitogenic anti-CD3 antibody. Blockade of RyR at initiation of culture or as late as 24 h after T cell receptor stimulation inhibited T cell proliferation, suggesting a requirement for sustained RyR signaling during cell cycle progression. Although flow cytometry revealed that RyR blockade had little effect on activation-induced expression of the alpha chain (CD25) of the high affinity IL-2 receptor, the inhibitory effect of RyR antagonists could not be reversed by the addition of exogenous IL-2 at initiation of culture. In addition, both ruthenium red and dantrolene had a strong inhibitory effect on IL-2-dependent proliferation of CTLL-2 T cells. These data indicate that RyR are involved in regulating IL-2 receptor signaling that drives T cell progression through the cell cycle. We conclude that RyR-associated Ca(++) signaling regulates T cell proliferation by promoting both IL-2 synthesis and IL-2-dependent cell cycle progression.     

Community outreach as an iterative dialogue among scientists and communities in the Texas gulf coast region

In response to significant environmental health challenges in Southeast Texas, a National Institute for Environmental Health Sciences Center at the University of Texas Medical Branch at Galveston was created to promote and conduct inter-disciplinary research in the areas of: (1) the molecular biology of DNA repair, replication and mutagenesis, (2) asthma pathogenesis in response to oxidative stress and viral exposures, and (3) environmental toxicant biotransformation. In addition, the NIEHS Center maintains close ties with neighboring communities through an active Community Outreach & Education Program (COEP) that develops and disseminates translational materials for use in environmental health awareness outreach, toxicology consultation, K-12 curriculum enrichment and in developing site-specific Community Partnership projects. The COEP core service divisions include: Environmental Arts & Sciences, Asthma Outreach & Education, Theater Outreach & Education, and Public Forum & Toxics Assistance. Public Forums focus on the use of Augusto Boal's Forum Theater dramaturgy to include the voices and local knowledge of communities within the process of Participatory Research. Forums create the preconditions for significant partnerships that link the hazardous risk perceptions and environmental health needs of communities with the expertise of NIEHS Center investigators and translational services provided through COEP outreach programs. The Forum process also creates leadership cores within environmentally challenged communities that facilitate the ongoing translational process and maintain the vital linkage between the health needs of communities and the analytic tools and the field and clinical technologies of the environmental sciences.     

A G577R mutation in the human AR P box results in selective decreases in DNA binding and in partial androgen insensitivity syndrome

We have characterized a novel mutation of the human AR, G577R, associated with partial androgen insensitivity syndrome. G577 is the first amino acid of the P box, a region crucial for the selectivity of receptor/DNA interaction. Although the equivalent amino acid in the GR (also Gly) is not involved in DNA interaction, the residue at the same position in the ER (Glu) interacts with the two central base pairs in the PuGGTCA motif. Using a panel of 16 palindromic probes that differ in these base pairs (PuGNNCA) in gel shift experiments with either the AR DNA-binding domain or the full length receptor, we observed that the G577R mutation does not induce binding to probes that are not recognized by the wild-type AR. However, binding to the four PuGNACA elements recognized by the wild-type AR was affected to different degrees, resulting in an altered selectivity of DNA response element recognition. In particular, AR-G577R did not interact with PuGGACA palindromes. Modeling of the complex between mutant AR and PuGNACA motifs indicates that the destabilizing effect of the mutation is attributable to a steric clash between the C beta of Arg at position 1 of the P box and the methyl group of the second thymine residue in the TGTTCPy arm of the palindrome. In addition, the Arg side chain can interact with G or T at the next position (PuGCACA and PuGAACA elements, respectively). The presence of C is not favorable, however, because of incompatible charges, abrogating binding to the PuGGACA element. Transactivation of several natural or synthetic promoters containing PuGGACA motifs was drastically reduced by the G577R mutation. These data suggest that androgen target genes may be differentially affected by the G577R mutation, the first natural mutation characterized that alters the selectivity of the AR/DNA interaction. This type of mutation may thus contribute to the diversity of phenotypes associated with partial androgen insensitivity syndrome.     

S-layer-coated liposomes as a versatile system for entrapping and binding target molecules

In the present study, unilamellar liposomes coated with the crystalline bacterial cell surface layer (S-layer) protein of Bacillus stearothermophilus PV72/p2 were used as matrix for defined binding of functional molecules via the avidin- or streptavidin-biotin bridge. The liposomes were composed of dipalmitoyl phosphatidylcholine, cholesterol and hexadecylamine in a molar ratio of 10:5:4 and they had an average size of 180 nm. For introducing specific functions into the S-layer lattice without affecting substances encapsulated within the liposomes, crosslinking and activation reagents had to be identified which did not penetrate the liposomal membrane. Among different reagents, a hydrophilic dialdehyde generated by periodate cleavage of raffinose and a sulfo-succinimide activated dicarboxylic acid were found to be impermeable for the liposomal membrane. Both reagents completely crosslinked the S-layer lattice without interfering with its regular structure. Biotinylation of S-layer-coated liposomes was achieved by coupling p-diazobenzoyl biocytin which preferably reacts with the phenolic residue of tyrosine or with the imidazole ring of histidine. By applying this method, two biotin residues accessible for subsequent avidin binding were introduced per S-layer subunit. As visualized by labeling with biotinylated ferritin, an ordered monomolecular layer of streptavidin was formed on the surface of the S-layer-coated liposomes. As a second model system, biotinylated anti-human IgG was attached via the streptavidin bridge to the biotinylated S-layer-coated liposomes. The biological activity of the bound anti-human IgG was confirmed by ELISA.     

Concordance of Epileptic Networks Associated with Epileptic Spikes Measured by High-Density EEG and Fast fMRI

Objective

The present study aims to investigate whether a newly developed fast fMRI called MREG (magnetic resonance encephalography) measures metabolic changes related to interictal epileptic discharges (IED). For this purpose BOLD changes are correlated with the IED distribution and variability.

Methods

Patients with focal epilepsy underwent EEG-MREG using a 64 channel cap. IED voltage maps were generated using 32 and 64 channels and compared regarding their correspondence to the BOLD response. The extents of IEDs (defined as number of channels with >50% of maximum IED negativity) were correlated with the extents of positive and negative BOLD responses. Differences in inter-spike variability were investigated between interictal epileptic discharges (IED) sets with and without concordant positive or negative BOLD responses.

Results

17 patients showed 32 separate IED types. In 50% of IED types the BOLD changes could be confirmed by another independent imaging method. The IED extent significantly correlated with the positive BOLD extent (p = 0.04). In 6 patients the 64-channel EEG voltage maps better reflected the positive or negative BOLD response than the 32-channel EEG; in all others no difference was seen. Inter-spike variability was significantly lower in IED sets with than without concordant positive or negative BOLD responses (with p = 0.04).

Significance

Higher density EEG and fast fMRI seem to improve the value of EEG-fMRI in epilepsy. The correlation of positive BOLD and IED extent could suggest that widespread BOLD responses reflect the IED network. Inter-spike variability influences the likelihood to find IED concordant positive or negative BOLD responses, which is why single IED analysis may be promising.     

Body temperature and respiratory dynamics in un-shaded beef cattle

In this study body temperature (BT, °C) and panting score (PS, 0–4.5; where 0?=?no panting/no stress and 4.5?=?catastrophic stress) data were obtained from 30 Angus steers housed outside over 120 days Steers were implanted with a BT transmitter on day ?31, BT was recorded at 30-min intervals to a data logger and downloaded each day to a database. The cattle were housed in ten outdoor un-shaded pens with an earthen floor, eight of which had a pen floor area of 144 m² (three transmitter steers plus five non-transmitter steers; 18 m²/steer) and two had an area of 168 m² (three transmitter steers and six non-transmitter steers; 18.7 m²/steer). Only data from the transmitter steers were used in this study. The PS of the steers was obtained daily (± 15 min) at 0600 hours (AM), 1200 hours (MD) and 1600 hours (PM). At the same times climate variables (ambient temperature, black globe temperature, solar radiation, relative humidity, wind speed and rainfall) were obtained from an on-site weather station. PS observations were made from outside the pens so as not to influence cattle responses. The two closest BT values to the time when PS was obtained were downloaded retrospectively from a logger and averaged. A total of 8,352 observations were used to generate second order polynomial response curves: (AM) y?=?39.08?+?0.009?x +?0.137x ² (R ²?=?0.94; P? y?=?39.09?+?0.914x ? 0.080x ² (R ²?=?0.89; P? y?=?39.52?+?0.790x ? 0.068x ² (R ²?=?0.83; P?x?PS. These data suggest that PS is a good indicator of body temperature. The BT at MD corresponded to slightly lower PS compared with PM, e.g., for PS 1; BT at MD?=?39.1?±?0.05 °C whereas BT at PM?=?39.5?±?0.05 °C. However during AM, BT was lower (P?相似文献