Inadvertent Propagation of Factor VII Deficiency in a Canine Mucopolysaccharidosis Type I Research Breeding Colony

Issues of cost and genetics can result in inbreeding of canine genetic disease colonies. Beagles often are used to maintain such colonies, providing stock for outcrosses. Factor VII (FVII) deficiency is a hemostatic disorder found at increased frequency in beagles and has been characterized at the DNA level. Deficiency of FVII presents obstacles in colonies founded with beagles. An initial finding of a FVII-deficient pup from a longstanding colony prompted us to evaluate FVII deficiency fully in this colony. Current and archival records and tissues were used to reconstruct the colony pedigree, assess the contribution from beagles, and test samples to document the source and frequency of the mutant FVII allele. As part of this study we developed a PCR-based diagnostic assay that was simpler than what was previously available. Pedigree analysis revealed a founder effect implicating beagles that led to high frequency (55%) of the mutant allele. In addition, affected animals were identified. The complete picture of the clinical effect within the colony remains unclear, but unusual neonatal presentations, including hemoabdomen, have occurred in pups affected with FVII deficiency. Use of a PCR-based diagnostic assay to screen all potential beagle breeding stock will prevent similar occurrences of FVII deficiency in future canine research colonies.Abbreviations: FVII, factor VII; MPS I, mucopolysaccharidosis I; PT, prothrombin timeThe importance of developing clinically relevant large animal models for human genetic diseases is becoming increasingly evident.⁴ For example, preclinical assessments of gene transfer experiments require large long-lived animal models physiologically and genetically comparable to humans. Canine models are ideal because their genome has been sequenced, they are large and long-lived, and because more than 60% of inherited diseases of dogs are known to be homologs of human diseases.⁴The maintenance of genetic diseases in research colonies results, for all practical purposes, in a founder effect by which allelic frequencies in a research colony may be skewed upward from those in the general population, due to founding of the colony by a limited number of animals. This founder effect is due to insufficient genetic outcrosses, resulting from economic constraints and considerations such as the inbreeding needed with a recessive condition. Practically speaking, colony founders may inevitably be incompletely characterized at the genetic level, potentially leading to increased prevalence of an additional genetic disease. When available, practical screening tests (clotting times, cardiac evaluation, and so on) or breed-specific genetic tests should be conducted to reduce additional genetic diseases within a research colony.The occurrence of additional genetic diseases in research colonies can limit or confound the primary research objective and affect the number of research animals used and their health and welfare. Inherited factor VII (FVII) deficiency in beagles is such a condition.⁸ Although largely an asymptomatic defect, this autosomal recessive hemostatic disorder, can lead to excessive bleeding after surgery or trauma, hematoma formation, body cavity bleeding, and persistent uterine and vaginal hemorrhage.²³ Factor VII deficiency also occurs in Alaskan malamutes,¹⁴ mixed breeds,¹³ and Alaskan klee kai dogs.¹¹ Clinical symptoms in canines can be reduced by transfusions with fresh plasma or blood, or administration of recombinant activated human FVII.^9,17 However, treatments are only a temporary solution, because the half-life of FVII protein is only 3 to 4 h and, in canines, treatment with human proteins raises concern about antibody responses to those proteins, thus potentially limiting further therapy. The FVII mutation initially described in beagles (referred to henceforth as the ‘beagle mutation’ in full recognition of its occurrence in additional breeds) is well described.¹ Furthermore the beagle mutation has been documented to cause the FVII deficiency seen in the Alaskan klee kai,¹¹ Airedale terrier, giant schnauzer, and Scottish deerhound.²² The published assay is a restriction digest to test beagles for the causative transitional missense mutation of a guanine-to-adenine located in exon 5 (leading to the G96E mutant protein).¹ However, because the assay relies on an enzyme with multiple sites in the resultant amplicon, interpretation of results can be problematic, potentially requiring direct DNA sequencing for confirmation of the genotype.Herein we present data from a long-standing canine research breeding colony for mucopolysaccharidosis type I (MPS I) which indicate that FVII deficiency should be a primary concern when developing research colonies by using beagle breeding stock. We suspected factor VII deficiency in this colony after an episode of hemoabdomen in a neonate, which was noted shortly after the colony was transferred to a different institution. Using an improved PCR-based diagnostic assay for the beagle FVII mutation, we have documented the history of this mutant allele within the colony in detail and have identified the presence of this allele in other canine colonies.     

Influencing hematopoietic differentiation of mouse embryonic stem cells using soluble heparin and heparan sulfate saccharides

Heparan sulfate proteoglycans (HSPG) encompass some of the most abundant macromolecules on the surface of almost every cell type. Heparan sulfate (HS) chains provide a key interaction surface for the binding of numerous proteins such as growth factors and morphogens, helping to define the ability of a cell to respond selectively to environmental cues. The specificity of HS-protein interactions are governed predominantly by the order and positioning of sulfate groups, with distinct cell types expressing unique sets of HS epitopes. Embryos deficient in HS-synthesis (Ext1(-/-)) exhibit pre-gastrulation lethality and lack recognizable organized mesoderm and extraembryonic tissues. Here we demonstrate that embryonic stem cells (ESCs) derived from Ext1(-/-) embryos are unable to differentiate into hematopoietic lineages, instead retaining ESC marker expression throughout embryoid body (EB) culture. However hematopoietic differentiation can be restored by the addition of soluble heparin. Consistent with specific size and composition requirements for HS:growth factor signaling, chains measuring at least 12 saccharides were required for partial rescue of hematopoiesis with longer chains (18 saccharides or more) required for complete rescue. Critically N- and 6-O-sulfate groups were essential for rescue. Heparin addition restored the activity of multiple signaling pathways including bone morphogenic protein (BMP) with activation of phospho-SMADs re-established by the addition of heparin. Heparin addition to wild-type cultures also altered the outcome of differentiation, promoting hematopoiesis at low concentrations, yet inhibiting blood formation at high concentrations. Thus altering the levels of HS and HS sulfation within differentiating ESC cultures provides an attractive and accessible mechanism for influencing cell fate.     

Glycosaminoglycans as regulators of stem cell differentiation

ES (embryonic stem) cell differentiation is dependent on the presence of HS (heparan sulfate). We have demonstrated that, during differentiation, the evolution of specific cell lineages is associated with particular patterns of GAG (glycosaminoglycan) expression. For example, different HS epitopes are synthesized during neural or mesodermal lineage formation. Cell lines mutant for various components of the HS biosynthetic pathway are selectively impaired in their differentiation, with lineage-specific effects observed for some lines. We have also observed that the addition of soluble GAG saccharides to cells, with or without cell-surface HS, can influence the pace and outcome of differentiation, again highlighting specific pattern requirements for particular lineages. We are combining this work with ongoing studies into the design of artificial cell environments where we have optimized three-dimensional scaffolds, generated by electrospinning or by the formation of hydrogels, for the culture of ES cells. By permeating these scaffolds with defined GAG oligosaccharides, we intend to control the mechanical environment of the cells (via the scaffold architecture) as well as their biological signalling environment (using the oligosaccharides). We predict that this will allow us to control ES cell pluripotency and differentiation in a three-dimensional setting, allowing the generation of differentiated cell types for use in drug discovery/testing or in therapeutics.     

Efficient Improvement of Silage Additives by Using Genetic Algorithms

The enormous variety of substances which may be added to forage in order to manipulate and improve the ensilage process presents an empirical, combinatorial optimization problem of great complexity. To investigate the utility of genetic algorithms for designing effective silage additive combinations, a series of small-scale proof of principle silage experiments were performed with fresh ryegrass. Having established that significant biochemical changes occur over an ensilage period as short as 2 days, we performed a series of experiments in which we used 50 silage additive combinations (prepared by using eight bacterial and other additives, each of which was added at six different levels, including zero [i.e., no additive]). The decrease in pH, the increase in lactate concentration, and the free amino acid concentration were measured after 2 days and used to calculate a “fitness” value that indicated the quality of the silage (compared to a control silage made without additives). This analysis also included a “cost” element to account for different total additive levels. In the initial experiment additive levels were selected randomly, but subsequently a genetic algorithm program was used to suggest new additive combinations based on the fitness values determined in the preceding experiments. The result was very efficient selection for silages in which large decreases in pH and high levels of lactate occurred along with low levels of free amino acids. During the series of five experiments, each of which comprised 50 treatments, there was a steady increase in the amount of lactate that accumulated; the best treatment combination was that used in the last experiment, which produced 4.6 times more lactate than the untreated silage. The additive combinations that were found to yield the highest fitness values in the final (fifth) experiment were assessed to determine a range of biochemical and microbiological quality parameters during full-term silage fermentation. We found that these combinations compared favorably both with uninoculated silage and with a commercial silage additive. The evolutionary computing methods described here are a convenient and efficient approach for designing silage additives.     

A Cell Culture Model for Androgen Effects in Motor Neurons

Abstract: Androgens are known to alter the morphology, survival, and axonal regeneration of lower motor neurons in vivo. To understand better the molecular mechanisms of androgen action in neurons, we created a model system by stably expressing the human androgen receptor (AR) in motor neuron hybrid cells. Motor neuron hybrid cells express markers consistent with anterior horn cells and can be differentiated into a neuronal phenotype. When differentiated in the presence of androgen, AR-expressing cells, but not control cells, exhibit a dose-dependent change in morphology: androgen-treated cells develop larger cell bodies and broader neuritic processes while continuing to express neuronal markers. In addition, androgen promotes the survival of AR-expressing cells, but not control cells, under low-serum conditions. Our results demonstrate a direct trophic effect of androgens on lower motor neurons, mediated through the AR expressed in this population of neurons.     

Viability of Cryptosporidium parvum during ensilage of perennial ryegrass

The survival of Cryptosporidium parvum during ensilage of perennial ryegrass was examined in laboratory silos with herbage prepared in one of three different ways; either untreated, inoculated with a strain of Lactobacillus plantarum or by direct acidification with formic acid. The pH values of all silages initially fell below 4.5, but only formic acid-treated silage remained stable at less than pH 4 after 106 d, with the pH of the untreated and inoculant-treated silages rising to above 6. The formic acid-treated silage had a high lactic acid concentration (109 g kg^-1 dry matter (DM)) and low concentrations of propionic and butyric acids after 106 d. However, the untreated and inoculant-treated silages showed an inverse relationship, with low lactic acid concentrations and high concentrations of acetic, propionic and butyric acids. These silages also contained ammonia-N concentrations in excess of 9 g kg^-1 DM. In terms of the viability of Cryptosporidium parvum oocysts very few differences were seen after 14 d of ensilage with ca 50% remaining viable, irrespective of treatment and total numbers had declined from the initial level of 5.9 × 10⁴ to 1 x 10⁴ g^-1 fresh matter. Total oocyst numbers remained approximately the same until the end of the ensiling period, with the percentage of viable oocysts declining to 46, 41 and 32% respectively for formic acid, inoculant and untreated silages. The results are discussed in terms of changes occurring during the silage fermentation, in particular the products which may influence the survival of Cryptosporidium and implications for agricultural practice and the health of silage fed livestock.     

Photovoltaic Materials and Devices Based on the Alloyed Kesterite Absorber (AgxCu1–x)2ZnSnSe4

The photovoltaic absorber Cu₂ZnSn(S_xSe_1–x)₄ (CZTSSe) has attracted interest in recent years due to the earth‐abundance of its constituents and the realization of high performance (12.6% efficiency). The open‐circuit voltage in CZTSSe devices is believed to be limited by absorber band tailing caused by the exceptionally high density of Cu/Zn antisites. By replacing Cu in CZTSSe with Ag, whose covalent radius is ≈15% larger than that of Cu and Zn, the density of I–II antisite defects is predicted to drop. The fundamental properties of the mixed Ag‐Cu kesterite compound are reported as a function of the Ag/(Ag + Cu) ratio. The extent of band tailing is shown to decrease with increasing Ag. This is verified by comparing the optical band gap extrapolated from transmission data with the position of the room‐temperature photoluminescence peak; these values converge for the pure‐Ag compound. Additionally, the pinning of the Fermi level in CZTSSe, attributed to heavy defect compensation and band tailing, is not observed in the pure‐Ag compound, offering further evidence of improved electronic structure. Finally, a device efficiency of 10.2% is reported for a device containing 10% Ag (no antireflection coating); this compares to ≈9% (avg) efficiency for the baseline pure‐Cu CZTSe.