Maximizing antibody production in a targeted integration host by optimization of subunit gene dosage and position

Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system.     

Sex-biased dispersal and the speed of two-sex invasions

Population models that combine demography and dispersal are important tools for forecasting the spatial spread of biological invasions. Current models describe the dynamics of only one sex (typically females). Such models cannot account for the sex-related biases in dispersal and mating behavior that are typical of many animal species. In this article, we construct a two-sex integrodifference equation model that overcomes these limitations. We derive an explicit formula for the invasion speed from the model and use it to show that sex-biased dispersal may significantly increase or decrease the invasion speed by skewing the operational sex ratio at the invasion's low-density leading edge. Which of these possible outcomes occurs depends sensitively on complex interactions among the direction of dispersal bias, the magnitude of bias, and the relative contributions of females and males to local population growth.     

Azadirachtin disrupts formation of organised microtubule arrays during microgametogenesis of Plasmodium berghei

Transmission of malaria parasites from vertebrate blood to the mosquito vector depends critically on the differentiation of the gametocytes into gametes. This occurs in response to environmental stimuli encountered by the parasite in the mosquito bloodmeal. Male gametogenesis involves three rounds of DNA replication and endomitosis, and the assembly de novo of 8 motile axonemes. Azadirachtin, a plant limnoid and insecticide with an unkown mode of action, specifically inhibits the release of motile gametes from activated microgametocytes but does not inhibit growth and replication of a sexual blood stages. We have combined confocal laser scanning microscopy and transmission electron microscopy to examine the effect of azadirachtin on the complex reorganisation of the microtubule cytoskeleton during gametogenesis in Plasmodium berghei. Neither the replication of the genome nor the ability of tubulin monomers to assemble into microtubules upon gametocyte activation were prevented by azadirachtin. However, the drug interfered with the formation of mitotic spindles and with the assembly of microtubules into typical axonemes. Our observations suggest that azadarachtin specifically disrupts the patterning of microtubules into more complex structures, such as mitotic spindles and axonemes.     

Health service accreditation: report of a pilot programme for community hospitals.

Voluntary accreditation in the United Kingdom is being used by health care providers to improve and market their services and by commissioners to define and monitor service contracts. In a three year pilot scheme in the south west of England, 43 out of 57 eligible community hospitals volunteered to be surveyed; 37 of them were ultimately accredited for up to two years by the hospital accreditation programme. The main causes for non-accreditation related to safety, clinical records, and medical organisation. Follow up visits in 10 hospitals showed that, overall, 69% of recommendations were implemented. An independent survey of participating hospitals showed the perceived benefits to include team building, review of operational policies, improvement of data systems, and the generation of local prestige. Purchasers are increasingly influenced by accreditation status but are mostly unwilling to finance the process directly. None the less, the concept may become an important factor moderating the quality of service in the new NHS.     

Intergroup variation in stable isotope ratios reflects anthropogenic impact on the Barbary macaques (Macaca sylvanus) of Gibraltar

Interactions with humans impact many aspects of behavior and ecology in nonhuman primates. Because of the complexities of the human–nonhuman primate interface, methods are needed to quantify the effects of anthropogenic interactions, including their intensity and differential impacts between nonhuman primate groups. Stable isotopes can be used to quickly and economically assess intergroup dietary variation, and provide a framework for the development of specific hypotheses about anthropogenic impact. This study uses stable carbon and nitrogen isotope analysis to examine intraspecific variation in diet between five groups of Barbary macaques, Macaca sylvanus, in the Upper Rock Nature Reserve, Gibraltar. Analysis of hair from 135 macaques showed significant differences in δ¹³C and δ¹⁵N values between a group with minimal tourist contact and groups that were main tourist attractions. Because we observed no overt physiological or substantial behavioral differences between the groups, feeding ecology is the most likely cause of any differences in stable isotope ratios. Haphazard provisioning by tourists and Gibraltarians is a likely source of dietary variation between groups. Stable isotope analysis and observational data facilitate a deeper understanding of the feeding ecology of the Barbary macaques relevant to the role of an anthropogenic ecology for the species.     

Morphologically cryptic biological species within the liverwort Frullania asagrayana

? Premise of the study: The Frullania tamarisci complex includes eight Holarctic liverwort species. One of these, F. asagrayana, is distributed broadly throughout eastern North America from Canada to the Gulf Coast. Preliminary genetic data suggested that the species includes two groups of populations. This study was designed to test whether the two groups are reproductively isolated biological species. ? Methods: Eighty-eight samples from across the range of F. asagrayana, plus 73 samples from one population, were genotyped for 13 microsatellite loci. Sequences for two plastid loci and nrITS were obtained from 13 accessions. Genetic data were analyzed using coalescent models and Bayesian inference. ? Key results: Frullania asagrayana is sequence-invariant at the two plastid loci and ITS2, but two clear groups were resolved by microsatellites. The two groups are largely reproductively isolated, but there is a low level of gene flow from the southern to the northern group. No gene flow was detected in the other direction. A local population was heterogeneous but displayed strong genetic structure. ? Conclusions: The genetic structure of F. asagrayana in eastern North America reflects morphologically cryptic differentiation between reproductively isolated groups of populations, near-panmixis within groups, and clonal propagation at local scales. Reproductive isolation between groups that are invariant at the level of nucleotide sequences shows that caution must be exercised in making taxonomic and evolutionary inferences from reciprocal monophyly (or lack thereof) between putative species.     

Environmental factors influencing zooplankton species composition of lakes in north-central Ontario,Canada

To assess the relative importance of lake chemistry, morphometry and zoogeography on limnetic zooplankton, we collected zooplankton, water, and morphometric data from 132 headwater Canadian Shield lakes in 6 regions across north-central Ontario. A subset of these lakes (n = 52) were fished with gill nets. We clustered lakes based on their zooplankton species composition (presence/absence). Discriminant analysis was employed to determine how well lake characteristics could predict zooplankton community types. Correct classification of zooplankton communities for three models ranged from 72 to 91%. Lake size, lake location, and buffering capacity were ranked as the most important factors separating lake groups. Fish abundance (CPUE) was not significant in distinguishing between zooplankton communities. Though the range of lake sizes was limited (1–110 ha), larger lakes tended to support more species. Lake location (zoogeography) also influenced species composition patterns. Although Algoma lakes tended to be larger (\-x = 18.0 ha, other lakes \-x = 2.5 ha), they supported relatively depauperate zooplankton communities. Buffering capacity was ranked third in the discriminant analysis models, but pH and alkalinity were not significantly different between lake groups.     

Prosthetic group content and ligand binding properties of a spinach catalase

A recently discovered form of spinach catalase that contains both a novel heme and protoheme as prosthetic groups has been characterized using immunological and spectroscopic techniques. The enzyme appears to be a dimer of identical Mr 60,000 monomers. Extraction of the non-covalently bound prosthetic groups, followed by thin-layer chromatography of the extract, suggested that the novel heme contains four carboxylic acid side-chain groups. The resonance Raman spectrum of the resting enzyme indicates that the protoheme prosthetic group is five-coordinate and high-spin. The enzyme was shown to bind formate, azide and cyanide. Cyanide and azide binding to catalase are biphasic, suggesting the existence of two different binding sites for cyanide and azide in the enzyme. Results obtained from EPR and resonance Raman spectroscopies also support the hypothesis that two different ligand-binding sites are present in the enzyme. Western blots suggest that the Mr 60,000 peptide of the novel heme-containing catalase is similar or identical to that of a previously characterized, exclusively protoheme-containing, tetrameric catalase.     

Diffusible factors are responsible for differences in nuclease sensitivity among chromatins originating from different cell types

We have examined the kinetics of nuclease digestion of chromatin from committed and uncommitted cells in experiments where the nuclei are mixed and co-digested. Cultures of the sea urchin, Arbacia punctulata, were grown to the 16-cell stage in either [3H]thymidine or [14C]thymidine and the macromere, mesomere, and micromere cell types separated. After isolation, sets of nuclei with two different blastomere types (each having different radionucleotide tagging) were mixed and co-digested with micrococcal nuclease or DNase. I. The extent of digestion was monitored by solubility in 5% perchloric acid (PCA). We find no significant differences in initial digestion rates or limit digests among the different cell types when co-digested with either nuclease. Differences in nuclease sensitivity observed when nuclei are digested separately are abolished when nuclei are probed in a mixing experiment. The results support the hypothesis that phenotypic differences in digestibility among different cell types in vitro reflect differences in chromatin-condensing factors which can diffuse between nuclei.     

An electron-capture gas–liquid-chromatographic method for the determination of prostaglandin F2α in biological fluids

A sensitive electron-capture gas-liquid-chromatographic method for the determination of sub-nanogram quantities of prostaglandin F(2alpha) was developed. The method is based on the sub-microgram scale conversion of the prostaglandin into the electron-capturing pentafluorobenzyl ester, and analysis of the latter as the tris-trimethylsilyl ether. The lower limit of detection was 12.5pg of the ester injected ;on-column' as the silylated product. The method was successfully applied to the determination of prostaglandin F(2alpha) in monkey plasma. The specificity of the analytical procedure was increased by incorporating a thin-layer chromatographic fractionation before gas-liquid chromatography. The utility of the analytical methodology developed was demonstrated by its application to the determination of plasma concentrations of intact prostaglandin F(2alpha) in a Rhesus monkey, after subcutaneous administration of a single dose of prostaglandin F(2alpha). The electron-capture gas-liquid-chromatographic assay is compared with the radioimmunoassay and the gas-liquid-chromatographic-mass-spectrometry assay for the determination of prostaglandin F(2alpha).