Metabolic variation in natural populations of wild yeast

Ecological diversification depends on the extent of genetic variation and on the pattern of covariation with respect to ecological opportunities. We investigated the pattern of utilization of carbon substrates in wild populations of budding yeast Saccharomyces paradoxus. All isolates grew well on a core diet of about 10 substrates, and most were also able to grow on a much larger ancillary diet comprising most of the 190 substrates we tested. There was substantial genetic variation within each population for some substrates. We found geographical variation of substrate use at continental, regional, and local scales. Isolates from Europe and North America could be distinguished on the basis of the pattern of yield across substrates. Two geographical races at the North American sites also differed in the pattern of substrate utilization. Substrate utilization patterns were also geographically correlated at local spatial scales. Pairwise genetic correlations between substrates were predominantly positive, reflecting overall variation in metabolic performance, but there was a consistent negative correlation between categories of substrates in two cases: between the core diet and the ancillary diet, and between pentose and hexose sugars. Such negative correlations in the utilization of substrate from different categories may indicate either intrinsic physiological trade‐offs for the uptake and utilization of substrates from different categories, or the accumulation of conditionally neutral mutations. Divergence in substrate use accompanies genetic divergence at all spatial scales in S. paradoxus and may contribute to race formation and speciation.     

Purification process development of a recombinant monoclonal antibody expressed in glycoengineered Pichia pastoris

A robust and scalable purification process was developed to quickly generate antibody of high purity and sufficient quantity from glycoengineered Pichia pastoris fermentation. Protein A affinity chromatography was used to capture the antibody from fermentation supernatant. A pH gradient elution was applied to the Protein A column to prevent antibody precipitation at low pH. Antibody from Protein A chromatography contained some product related impurities, which were the misassembling of cleaved heavy chain, heavy chain and light chain. It also had some process related impurities, including Protein A residues, endotoxin, host cell DNA and proteins. Cation exchange chromatography with optimal NaCl gradient at pH 4.5-6.0 efficiently removed these product and process related impurities. The antibody from glycoengineered P. pastoris was comparable to its commercial counterpart in heterotetramer folding, physical stability and binding affinity.     

Low Bone Turnover and Low BMD in Down Syndrome: Effect of Intermittent PTH Treatment

Trisomy 21 affects virtually every organ system and results in the complex clinical presentation of Down syndrome (DS). Patterns of differences are now being recognized as patients' age and these patterns bring about new opportunities for disease prevention and treatment. Low bone mineral density (BMD) has been reported in many studies of males and females with DS yet the specific effects of trisomy 21 on the skeleton remain poorly defined. Therefore we determined the bone phenotype and measured bone turnover markers in the murine DS model Ts65Dn. Male Ts65Dn DS mice are infertile and display a profound low bone mass phenotype that deteriorates with age. The low bone mass was correlated with significantly decreased osteoblast and osteoclast development, decreased bone biochemical markers, a diminished bone formation rate and reduced mechanical strength. The low bone mass observed in 3 month old Ts65Dn mice was significantly increased after 4 weeks of intermittent PTH treatment. These studies provide novel insight into the cause of the profound bone fragility in DS and identify PTH as a potential anabolic agent in the adult low bone mass DS population.     

Responses of Dendroctonus brevicomis (Coleoptera: Curculionidae) in behavioral assays: implications to development of a semiochemical-based tool for tree protection

Currently, techniques for managing western pine beetle, Dendroctonus brevicomis LeConte (Coleoptera: Curculionidae, Scolytinae), infestations are limited to tree removals (thinning) that reduce stand density and presumably host susceptibility, and/or the use of insecticides to protect individual trees. There continues to be significant interest in developing an effective semiochemical-based tool for protecting trees from D. brevicomis attack, largely as an alternative to conventional insecticides. The responses of D. brevicomis to tree volatiles and verbenone were documented in eight experiments (trapping assays) conducted over a 4-yr period in which 88,942 individuals were collected. Geraniol, a tree volatile unique to Pinus ponderosa that elicits female-specific antennal responses in D. brevicomis, did not affect D. brevicomis behavior. Blends of two green leaf alcohols [hexanol + (Z)-3-hexen-1-ol] tested at two release rates (5.0 and 100.0 mg/d) had no effect on the response of D. brevicomis to attractant-baited traps. A nine-component blend [benzaldehyde, benzyl alcohol, guaiacol, nonanal, salicylaldehyde, (E)-2-hexenal, (E)-2-hexen-1-ol, (Z)-2-hexen-1-ol, and (-) -verbenone; NAVV] and subsequent revisions of this blend disrupted the response of D. brevicomis to attractant-baited traps in all experiments. The inhibitory effect of a revised five-component blend [nonanal, (E)-2-hexenal, (E)-2-hexen-1-ol, (Z)-2-hexen-1-ol, and (-)-verbenone; NAVV5] on the response of mountain pine beetle, D. ponderosae Hopkins, to attractant-baited traps was also documented. Acetophenone significantly reduced D. brevicomis attraction, but was not as effective as verbenone alone. Acetophenone increased the effectiveness of NAVV5 in one of two experiments. Furthermore, by adding acetophenone to NAVV5 we were able to remove the aldehydes from NAVV5 without compromising effectiveness, resulting in a novel four-component blend [acetophenone, (E)-2-hexen-1-ol + (Z)-2-hexen-1-ol, and (-)-verbenone; Verbenone Plus]. We discuss the implications of these and other results to development of Verbenone Plus as a semiochemical-based tool for management of D. brevicomis and D. ponderosae infestations.     

Identification of the orthopoxvirus p4c gene,which encodes a structural protein that directs intracellular mature virus particles into A-type inclusions

The orthopoxvirus gene p4c has been identified in the genome of the vaccinia virus strain Western Reserve. This gene encodes the 58-kDa structural protein P4c present on the surfaces of the intracellular mature virus (IMV) particles. The gene is disrupted in the genome of cowpox virus Brighton Red (BR), demonstrating that although the P4c protein may be advantageous for virus replication in vivo, it is not essential for virus replication in vitro. Complementation and recombination analyses with the p4c gene have shown that the P4c protein is required to direct the IMV into the A-type inclusions (ATIs) produced by cowpox virus BR. The p4c gene is highly conserved among most members of the orthopoxvirus genus, including viruses that produce ATIs, such as cowpox, ectromelia, and raccoonpox viruses, as well as those such as variola, monkeypox, vaccinia, and camelpox viruses, which do not. The conservation of the p4c gene among the orthopoxviruses, irrespective of their capacities to produce ATIs, suggests that the P4c protein provides functions in addition to that of directing IMV into ATIs. These findings, and the presence of the P4c protein in IMV but not extracellular enveloped virus (D. Ulaeto, D. Grosenbach, and D. E. Hruby, J. Virol. 70:3372-3377, 1996), suggest a model in which the P4c protein may play a role in the retrograde movement of IMV particles, thereby contributing to the retention of IMV particles within the cytoplasm and within ATIs when they are present. In this way, the P4c protein may affect both viral morphogenesis and processes of virus dissemination.     

Heterologous human immunodeficiency virus type 1 priming-boosting immunization strategies involving replication-defective adenovirus and poxvirus vaccine vectors

We compared the human immunodeficiency virus type 1 (HIV-1)-specific cellular immune responses elicited in nonhuman primates by HIV-1 gag-expressing replication-defective adenovirus serotype 5 (Ad5) or poxvirus vectors, used either alone or in combination with each other. The responses arising from a heterologous Ad5 priming-poxvirus boosting regimen were significantly greater than those elicited by homologous regimens with the individual vectors or by a heterologous poxvirus priming-Ad5 boosting regimen. The heterologous Ad5 priming-poxvirus boosting approach may have potential utility in humans as a means of inducing high levels of cellular immunity.     

The effects of low level of feeding on response to synchronization of estrus, ovulation rate and embryo loss in goats

Mature nonlactating British Saanen and Toggenburg does with a body score 2 were fed 25% (n=24) and 100% (n=16) maintenance rations from about 19 days before mating until slaughter at approximately 60 days after mating. Estrus was synchronized using PGF(2alpha), and the ovulation rate was determined by laparoscopic examination of the ovaries once between Days 6 and 10 after mating. Pregnancy rate, potential kidding rate and embryo loss were determined by counts of viable fetuses at slaughter. The proportion of does in estrus within 96 hours of PGF(2alpha) administration was not different (P<0.5) between the feed-restricted and the maintenance groups (71.0% and 87.5%, respectively); however, the time of onset of estrus after PGF(2alpha) tended to be longer (P=0.12) in the feed-restricted group. Ovulation rate, incidence of multiple ovulations and proportion of does pregnant at 60 days were significantly lower (P=0.0004, P=0.025, P=0.05, respectively) in the restricted group. More embryos from single than multiple ovulations were lost in the restricted group (P=0.01). There was no difference in the overall ovulatory activity between right and left ovaries in the 2 groups. Transuterine migrations were observed in all does that had unilateral multiple ovulations. No migration was observed in does which had single ovulations. These data indicate that restricted feed intake in goats tended to delay the onset of estrus and lowered the ovulation rate, incidence of multiple ovulations, and pregnancy rate.     

Links between parental life histories of wild salmon and the telomere lengths of their offspring

The importance of parental contributions to offspring development and subsequent performance is self‐evident at a genomic level; however, parents can also affect offspring fitness by indirect genetic and environmental routes. The life history strategy that an individual adopts will be influenced by both genes and environment; and this may have important consequences for offspring. Recent research has linked telomere dynamics (i.e., telomere length and loss) in early life to future viability and longevity. Moreover, a number of studies have reported a heritable component to telomere length across a range of vertebrates, although the effects of other parental contribution pathways have been far less studied. Using wild Atlantic salmon with different parental life histories in an experimental split‐brood in vitro fertilization mating design and rearing the resulting families under standardized conditions, we show that there can be significant links between parental life history and offspring telomere length (studied at the embryo and fry stage). Maternal life history traits, in particular egg size, were most strongly related to offspring telomere length at the embryonic stage, but then became weaker through development. In contrast, paternal life history traits, such as the father's growth rate in early life, had a greater association in the later stages of offspring development. However, offspring telomere length was not significantly related to either maternal or paternal age at reproduction, nor to paternal sperm telomere length. This study demonstrates both the complexity and the importance of parental factors that can influence telomere length in early life.     

Environmental DNA Marker Development with Sparse Biological Information: A Case Study on Opossum Shrimp (Mysis diluviana)

The spread of Mysis diluviana, a small glacial relict crustacean, outside its native range has led to unintended shifts in the composition of native fish communities throughout western North America. As a result, biologists seek accurate methods of determining the presence of M. diluviana, especially at low densities or during the initial stages of an invasion. Environmental DNA (eDNA) provides one solution for detecting M. diluviana, but building eDNA markers that are both sensitive and species-specific is challenging when the distribution and taxonomy of closely related non-target taxa are poorly understood, published genetic data are sparse, and tissue samples are difficult to obtain. To address these issues, we developed a pair of independent eDNA markers to increase the likelihood of a positive detection of M. diluviana when present and reduce the probability of false positive detections from closely related non-target species. Because tissue samples of closely-related and possibly sympatric, non-target taxa could not be obtained, we used synthetic DNA sequences of closely related non-target species to test the specificity of eDNA markers. Both eDNA markers yielded positive detections from five waterbodies where M. diluviana was known to be present, and no detections in five others where this species was thought to be absent. Daytime samples from varying depths in one waterbody occupied by M. diluviana demonstrated that samples near the lake bottom produced 5 to more than 300 times as many eDNA copies as samples taken at other depths, but all samples tested positive regardless of depth.