A natively paired antibody library yields drug leads with higher sensitivity and specificity than a randomly paired antibody library

Deep sequencing and single-chain variable fragment (scFv) yeast display methods are becoming more popular for discovery of therapeutic antibody candidates in mouse B cell repertoires. In this study, we compare a deep sequencing and scFv display method that retains native heavy and light chain pairing with a related method that randomly pairs heavy and light chain. We performed the studies in a humanized mouse, using interleukin 21 receptor (IL-21R) as a test immunogen. We identified 44 high-affinity binder scFv with the native pairing method and 100 high-affinity binder scFv with the random pairing method. 30% of the natively paired scFv binders were also discovered with the randomly paired method, and 13% of the randomly paired binders were also discovered with the natively paired method. Additionally, 33% of the scFv binders discovered only in the randomly paired library were initially present in the natively paired pre-sort library. Thus, a significant proportion of “randomly paired” scFv were actually natively paired. We synthesized and produced 46 of the candidates as full-length antibodies and subjected them to a panel of binding assays to characterize their therapeutic potential. 87% of the antibodies were verified as binding IL-21R by at least one assay. We found that antibodies with native light chains were more likely to bind IL-21R than antibodies with non-native light chains, suggesting a higher false positive rate for antibodies from the randomly paired library. Additionally, the randomly paired method failed to identify nearly half of the true natively paired binders, suggesting a higher false negative rate. We conclude that natively paired libraries have critical advantages in sensitivity and specificity for antibody discovery programs.     

Periaxonal and nodal plasticities modulate action potential conduction in the adult mouse brain

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A deletion mutation in GDF9 in sisters with spontaneous DZ twins.

A loss of function mutation in growth differentiation factor 9 (GDF9) in sheep causes increased ovulation rate and infertility in a dosage-sensitive manner. Spontaneous dizygotic (DZ) twinning in the human is under genetic control and women with a history of DZ twinning have an increased incidence of multiple follicle growth and multiple ovulation. We sequenced the GDF9 coding region in DNA samples from 20 women with DZ twins and identified a four-base pair deletion in GDF9 in two sisters with twins from one family. We screened a further 429 families and did not find the loss of function mutation in any other families. We genotyped eight single nucleotide polymorphisms across the GDF9 locus in 379 families with two sisters who have both given birth to spontaneous DZ twins (1527 individuals) and 226 triad families with mothers of twins and their parents (723 individuals). Using case control analysis and the transmission disequilibrium test we found no evidence for association between common variants in GDF9 and twinning in the families. We conclude that rare mutations in GDF9 may influence twinning, but twinning frequency is not associated with common variation in GDF9.     

Late-gestation ventricular myocardial reduction in fetuses of hyperglycemic CD1 mice is associated with increased apoptosis

BACKGROUND : Previous work in our laboratory showed reduced myocardium and dilated ventricular chambers in gestation day (GD) 17 hearts that were collected from hyperglycemic CD1 mouse dams. Pre-breeding maternal immune stimulation, using Freund's complete adjuvant (FCA), diminished the severity of these fetal heart lesions. The following experiments were performed to detect possible changes in fetal heart apoptotic cell death, under hyperglycemic conditions and with or without maternal immune stimulation. METHODS : Female CD1 mice were injected with 200 mg/kg of streptozocin (STZ) to induce insulin-dependent diabetes mellitus. Half of these mice received prior FCA injection. Fetal hearts were collected on GD 17 and myocardial apoptotic cells were quantified using flow cytometry. A panel of apoptosis regulatory genes (Bcl2, p53, Casp3, Casp9, PkCe) was then examined in the fetal myocardium using RT-PCR. RESULTS : Early apoptotic cells and late apoptotic/necrotic cells were significantly increased in fetal hearts from STZ or STZ+FCA dams. Pre-treatment with FCA reduced late apoptotic/necrotic cells to control level, suggesting some cell death protection was rendered by FCA. Paradoxically in the face of such increased cell death, the expression of pro-apoptotic genes Casp3 and Casp9 was decreased by diabetes, while the anti-apoptotic gene Bcl2 was increased. CONCLUSIONS : Maternal hyperglycemia causes dys-regulated apoptosis of fetal myocardial cells. Such effect may be prevented by maternal immune stimulation. Birth Defects Res (Part B) 86:409–415, 2009. © 2009 Wiley-Liss, Inc.     

Extent of digging and its possible underlying causal factors in penned blue foxes

A recent European animal welfare recommendation stresses the importance of studying digging behaviour in farm-born blue foxes (Alopex lagopus). The current study was conducted (1) to clarify the extent of digging and (2) to evaluate factors that motivate digging. In experiment 1, six juvenile male blue foxes were housed together from August to the following June in an earthen enclosure. Experiment 2 was conducted from July to December, using ten enclosures each containing two juvenile male blue foxes. Behaviour was monitored by 24-h video recordings and visual observations. Progress of digging was also followed by making scale drawings of all digging marks on paper. As early as the first study day, clear signs of digging were observed. Digging sites were concentrated below and close to nestboxes and pen walls. Maximally about 20% of the total enclosure area was affected. The total surface area of digging sites did not increase from late summer onwards because foxes tended simultaneously to cover part of the old sites when digging new ones. Motivational tendency to dig varied with time. Digging activity decreased during autumn and almost totally ceased during winter. In May, foxes resumed digging activity. Digging motivation was evaluated by two means: (1) by analyzing digging purpose (experiments 1 and 2), and (2) by the damming-up test (experiment 1), that is, after 10 months foxes that had been exposed to the earthen floor were transferred for 12 days into wire-mesh cages with no possibility to dig in the ground. Thereafter, foxes were transferred back into the earthen enclosure to measure the rebound of digging following deprivation. Foxes were observed to dig for the following reasons: (1) to make a hole or a resting site, (2) to locate an escape route, (3) to cache food, faeces, or sticks, (4) in response to a novel object (new nestbox, replacement of nestbox), and (5) displacement without any clear goal. Daily time spent digging averaged 7 min and 17 min per fox in Exps. 1 and 2, respectively. A clear rebound effect for digging was not identified. It can be concluded that digging is a complex behavioural pattern caused by a variety of motivations that can vary over time. The present study was unable to show unambiguously that digging is an important need for farmed foxes. Received: 28 February 2000 / Received in revised form: 6 June 2000 / Accepted: 20 June 2000     

THE EFFECTS OF SELECTED HERBICIDES ON ZYGOSPORE GERMINATION AND GROWTH OF CHLAMYDOMONAS MOEWUSII (CHLOROPHYCEAE,VOLVOCALES)11

A laboratory study was conducted, to examine and compare the sensitivity of vegetative cells and zygospores of Chlamydomonas moewusii Gerloff to 20 different herbicides. Under the culture conditions employed, both vegetative growth and zygospore germination were affected by certain herbicides and not by others. Over a concentration range from 1.0–80.0 μM, growth was inhibited to various degrees by herbicides containing ametryne, paraquat, endothall, diquat, diuron, linuron, propanil, dinoseb, ioxynil, atrzine, prometon, and alachlor. Zygospore germination was inhibited significantly by herbicides containing dinoseb, endothall, parquet, diquat, propanil, linuron, ioxynil, ametryne, fenac and picloram at 80.0 μM concentrations. Comparisons of the results obtained indicate that concentrations of herbicides which affect growth may or may not effect zygospore germination and vice versa. Zygospores may be more resistant than vegetative cells to some but not all herbicides.