In vivo transgenic mutation assays

Transgenic rodent gene-mutation models provide relatively quick and statistically reliable assays for gene mutations in the DNA from any tissue. This report summarizes those issues that have been agreed upon at a previous IWGT meeting [Environ. Mol. Mutagen. 35 (2000) 253], and discusses in depth those issues for which no consensus was reached before. It was previously agreed that for regulatory applications, assays should be based upon neutral genes, be generally available in several laboratories, and be readily transferable. For phage-based assays, five to ten animals per group should be analyzed, assuming a spontaneous mutant frequency (MF) of approximately 3x10(-5) mutants/locus and 125,000-300,000 plaque or colony forming units (pfu or cfu) per tissue per animal. A full set of data should be generated for a vehicle control and two dose groups. Concurrent positive control animals are only necessary during validation, but positive control DNA must be included in each plating. Tissues should be processed and analyzed in a blocked design, where samples from negative control, positive control and each treatment group are processed together. The total number of pfus or cfus and the MF for each tissue and animal are reported. Statistical tests should consider the animal as the experimental unit. Nonparametric statistical tests are recommended. A positive result is a statistically significant dose-response and/or statistically significant increase in any dose group compared to concurrent negative controls using an appropriate statistical model. A negative result is a statistically non-significant change, with all mean MFs within two standard deviations of the control. During the current workshop, a general protocol was agreed in which animals are treated daily for 28 consecutive days and tissues sampled 3 days after the final treatment. This recommendation could be modified by reducing or increasing the number of treatments or the length of the treatment period, when scientifically justified. Normally male animals alone are sufficient and normally at least one rapidly proliferating and one slowly proliferating tissue should be sampled. Although, as agreed previously, sequencing data are not normally required, they might provide useful additional information in specific circumstances, mainly to identify and correct for clonal expansion and in some cases to determine a mechanism associated with a positive response.     

Mucosal challenge of Macaca nemestrina with simian immunodeficiency virus (SIV) following SIV nucleocapsid mutant DNA vaccination

A simian immunodeficiency virus (SIV)(Mne) DNA clone was constructed that produces viruses containing a four amino acid deletion in the second zinc finger of the nucleocapsid (NC) domain of the Gag polyprotein. Viruses produced from this clone, although non-infectious both in vitro and in vivo, complete a majority of the steps in a single retroviral infection cycle. Eight pig-tailed macaques (Macaca nemestrina) were inoculated intramuscularly and subcutaneously three times over the course of 24 weeks with the NC mutant expressing DNA. These macaques, and four controls, were then challenged mucosally (intrarectally) with the homologous virus (SIV Mne CL E11S) and monitored for evidence of infection and clinical disease. Prior to challenge, a measurable humoral immune response was noted in four of eight immunized macaques. After challenge, all 12 macaques became infected, although four immunized animals greatly restricted their viral replication, and one immunized animal that controlled replication remains antibody negative. No disease has been evidence during the 46-week period of monitoring after challenge.     

Discovery of a vorapaxar analog with increased aqueous solubility

An analog of the thrombin receptor antagonist vorapaxar (SCH 530348) with increased aqueous solubility, compound 9c (SCH 602539), was discovered through incorporation of polar substituents on the pyridine ring of the himbacine-derived lead series. This analog retained the excellent potency, pharmacokinetic and safety properties of vorapaxar while increasing the aqueous solubility by 20-fold. Also presented are in vivo evaluations of this compound in a cynomolgus monkey platelet aggregation assay and in a Folts model of thrombosis in anesthetized monkeys.     

Activation-induced cytidine deaminase targets DNA at sites of RNA polymerase II stalling by interaction with Spt5

Activation-induced cytidine deaminase (AID) initiates antibody gene diversification by creating U:G mismatches. However, AID is not specific for antibody genes; Off-target lesions can activate oncogenes or cause chromosome translocations. Despite its importance in these transactions little is known about how AID finds its targets. We performed an shRNA screen to identify factors required for class switch recombination (CSR) of antibody loci. We found that Spt5, a factor associated with stalled RNA polymerase II (Pol II) and single stranded DNA (ssDNA), is required for CSR. Spt5 interacts with AID, it facilitates association between AID and Pol II, and AID recruitment to its Ig and non-Ig targets. ChIP-seq experiments reveal that Spt5 colocalizes with AID and stalled Pol II. Further, Spt5 accumulation at sites of Pol II stalling is predictive of AID-induced mutation. We propose that AID is targeted to sites of Pol II stalling in part via its association with Spt5.     

Demographic factors associated with prevalence of antibody to Sin Nombre virus in deer mice in the western United States

We used long-term data collected for up to 10 yr (1994-2004) at 23 trapping arrays (i.e., webs and grids) in Arizona, Colorado, Montana, and New Mexico to examine demographic factors known or suspected to be associated with risk of infection with Sin Nombre virus (SNV) in its natural host, the deer mouse (Peromyscus maniculatus). Gender, age (mass), wounds or scars, season, and local relative population densities were statistically associated with the period prevalence of antibody (used as a marker of infection) to SNV in host populations. Nevertheless, antibody prevalence and some of the risk factors associated with antibody prevalence, such as relative population density, gender bias, and prevalence of wounding, varied significantly among sites and even between nearby trapping arrays at a single site. This suggests that local microsite-specific differences play an important role in determining relative risk of infection by SNV in rodents and, consequently, in humans. Deer mouse relative population density varied among sites and was positively and statistically associated with infection prevalence, an association that researchers conducting shorter-term studies failed to demonstrate. Both wounding and antibody prevalence increased with mass class in both males and females; this increase was much more pronounced in males than in females and wounding was more frequent in adult males than in adult females. Prevalence of wounding was greatest among seropositive deer mice, regardless of mass class, but many deer mice without detectable wounds or scars eventually became infected. Many of these patterns, which will be useful in the development of predictive models of disease risk to humans, were only detected through the application of data collected over a long (10-yr) period and with abundant replication.     

Quantifying division of labor: borrowing tools from sociology,sociobiology, information theory,landscape ecology,and biogeography

How do we quantify division of labor? We review several fields (sociology, landscape ecology, statistics, information theory, and biogeography) that have been cognizant of these questions and been somewhat successful at answering them. We review fourteen indices for quantifying division of labor, sensu lato, which can be categorized into four families: Shannon’s index/entropy, Simpson’s index, geometric mean, and standard/absolute deviation (including coefficients of variation). We argue that those indices using matrix inputs will simultaneously quantify the interplay between all individuals and all tasks and will thus best capture the essence of division of labor. Received 29 March 2006; revised 16 November 2006; accepted 12 February 2007.     

Long-term hypoxia modulates expression of key genes regulating adrenomedullary function in the late gestation ovine fetus

We previously communicated that long-term hypoxia (LTH) resulted in a selective reduction in plasma epinephrine following acute stress in fetal sheep. The present study tested the hypothesis that LTH selectively reduces adrenomedullary expression of phenylethanolamine-N-methyltransferase (PNMT), the rate-limiting enzyme for epinephrine synthesis. We also examined the effect of LTH on adrenomedullary nicotinic, muscarinic, and glucocorticoid receptor (GR) expression. Ewes were maintained at high altitude (3,820 m) from 30 to 138 days gestation (dGA); adrenomedullary tissue was collected from LTH and age-matched, normoxic control fetuses at 139-141 dGA. Contrary to our hypothesis, in addition to PNMT, adrenomedullary expression (mRNA, protein) of tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) were reduced in the LTH fetus. Immunocytochemistry indicated that TH and DBH expression was lower throughout the medulla, while PNMT appeared to reflect a reduction in PNMT-expressing cells. Nicotinic receptor alpha 1, 2, 3, 5, 6, 7, beta 1, 2, and 4 subunits were expressed in the medulla of LTH and control fetuses. Messenger RNA for alpha 1 and 7 and beta 1 and 2 subunits was lower in LTH fetuses. Muscarinic receptors M1, M2, and M3 as well as the GR were also expressed, and no differences were noted between groups. In summary, LTH in fetal sheep has a profound effect on expression of key enzymes mediating adrenomedullary catecholamine synthesis. Further, LTH impacts nicotinic receptor subunit expression potentially altering cholinergic neurotransmission within the medulla. These findings have important implications regarding fetal cardiovascular and metabolic responses to stress in the LTH fetus.