Statistical convenience vs biological insight: consequences of data transformation for the analysis of fitness variation in heterogeneous environments

In plants, more favourable environmental conditions can lead to dramatic increases in both mean fitness and variance in fitness. This results in data that violate the equality-of-variance assumption of anova, a problem that most empiricists would address by log-transforming fitness values. Using heuristic data sets and simple simulations, we show that anova on log-transformed fitness consistently fails to match the outcome of selection in a heterogeneous environment or its sensitivity to environmental frequency. Only anova based on relative fitness within environments accurately predicts the sensitivity of genotype selection to the frequency of alternative environments. Parallel analyses of variance based on absolute fitness and relative fitness can bracket the expected success of alternative genotypes under hard and soft selection, respectively. For example, for Sinapis arvensis growing in full sun and partial shade treatments, families achieving high fitness in the best environment are favoured under hard selection, whereas soft selection favours different families that achieve consistently good performance across environments. Based on these findings, we recommend that log-transformation of fitness should no longer be standard practice in ecological genetics studies. Weighted anova is a preferable method for dealing with unequal variances, and investigators should also make greater use of techniques such as quantile regression or resampling to describe and evaluate fitness variation across heterogeneous environments.     

A selective, slow binding inhibitor of factor VIIa binds to a nonstandard active site conformation and attenuates thrombus formation in vivo

The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF.FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF.FVIIa (Ki = 0.35 +/- 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF.FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa.G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln217-Gly219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys192-Gly193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by approximately 70% at 0.4 mg/kg + 0.1 mg/kg/min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.     

Chronobiological factors for compassion satisfaction and fatigue among ambulatory oncology caregivers

Primary caregivers for victims of chronic illness and or trauma experience both positive and negative emotional consequences. These are broadly classified as compassion satisfaction (CS) and compassion fatigue (CF). Because one of the components of CF, burnout, varies with chronotype and sleep quality, we assessed the influence of chronobiological features on the broader constructs of CS and CF. Responses from primary ambulatory care oncology staff working dayshifts were assessed for potential relationships of chronotype and sleep quality with CS and CF using the professional quality of life scale. These were analyzed further in a multivariate model that included personality and job satisfaction as cofactors. We found that sleep quality was a key contributor to CS development and CF reduction. Morningness was positively linked to CS, but the univariate association was masked in the multivariate model. Job satisfaction (contingent rewards, nature of work and operating procedures) heavily influenced CS and CF development. Agreeableness and openness showed positive correlations with CS and negative with burnout, while emotional stability was linked to reduced CF. While job satisfaction and personality predictably played roles in the development of CS and CF, sleep quality and chronotype contributed significantly to benefits and negative consequences of providing care.     

While K-ras is essential for mouse development, expression of the K-ras 4A splice variant is dispensable

In mammals, the three classical ras genes encode four highly homologous proteins, N-Ras, H-Ras, and the isoforms K-Ras 4A and 4B. Previous studies have shown that K-ras is essential for mouse development and that while K-ras 4A and 4B are expressed during development, K-ras 4A expression is regulated temporally and spatially and occurs in adult kidney, intestine, stomach, and liver. In the present study, the pattern of K-ras 4A expression was examined in a wide range of wild-type adult mouse tissues, and gene targeting was used to generate K-ras 4A-deficient mice to examine its role in development. It was found that K-ras 4A is also expressed in uterus, lung, pancreas, salivary glands, seminal vesicles, bone marrow cells, and cecum, where it was the major K-Ras isoform expressed. Mating between K-ras(tmDelta4A/+) mice produced viable K-ras(tmDelta4A/tmDelta4A) offspring with the expected Mendelian ratios of inheritance, and these mice expressed the K-ras 4B splice variant only. K-ras(tmDelta4A/tmDelta4A) mice were fertile and showed no histopathological abnormalities on inbred (129/Ola) or crossbred (129/Ola x C57BL/6) genetic backgrounds. The results demonstrate that K-Ras 4A, like H- and N-Ras, is dispensable for normal mouse development, at least in the presence of functional K-Ras 4B.     

Enhanced production of mouse hybridomas to picomoles of antigen using EL-4 conditioned media with an in vitro immunization protocol

Summary We report here that the use of murine thymoma cell EL-4 conditioned medium enhances hybridoma yield in a low-antigen dose in vitro immunization protocol. This improved protocol allowed the production of a panel of monoclonal antibodies toDrosophila yolk proteins using less than 1 nanomole of antigen. We believe this refinement will be valuable for the application of hybridoma technology to biologically active materials that are hard to isolate and purify due to their low concentration in the biological fluids. This research was supported by the College of Agriculture and Life Sciences, University of Maryland, USDA-University of Maryland Cooperative Editor's Statement This observation should simplify in vitro immunization approaches and shed new light on the factors required for the in vitro immune response. Wallace L. McKeehan     

Calmodulin activity in yeast and mycelial phases of Ceratocystis ulmi

Abstract The yeast and mycelial phases of Ceratocystis ulmi contained roughly equivalent levels of calmodulin activity as determined by their ability to stimulate calmodulin-deficient bovine brain cAMP phosphodiesterase. This stimulation was calcium-dependent and could be inhibited by either dibucaine or trifluoperazine. Also, the concentration of dibucaine necessary to achieve the mycelium-to-yeast morphological conversion was found to be 3-fold greater in the presence of exogenous calcium. A model is presented in which only 30% of the cellular calmodulin need be complexed with calcium ions for mycelial development.     

Effects of an anthropogenic diet on indicators of physiological challenge and immunity of white ibis nestlings raised in captivity

When wildlife forage and/or live in urban habitats, they often experience a shift in resource availability and dietary quality. Some species even use human handouts, such as bread, as well as human refuse, as a large part of their new diets; yet the influences of this nutritional shift on health and survival remain unclear. American white ibises are increasingly being seen in urban areas in Florida; they collect handouts, such as bread and other food items, from humans in parks, and are also found foraging on anthropogenic sources in trash heaps. We hypothesized that the consumption of these new anthropogenic food sources may trigger increases in indicators of physiological challenge and dampen immune responses. We tested this experimentally by raising 20 white ibis nestlings in captivity, and exposing 10 to a simulated anthropogenic diet (including the addition of white bread and a reduction in seafood content) while maintaining 10 on a diet similar to what ibises consume in more natural environments. We then tested two indicators of physiological challenge (corticosterone and heat shock protein 70), assessed innate immunity in these birds via bactericidal assays and an in vitro carbon clearance assay, and adaptive immunity using a phytohemagglutinin skin test. The anthropogenic diet depressed the development of the ability to kill Salmonella paratyphi in culture. Our results suggest that consuming an anthropogenic diet may be detrimental in terms of the ability to battle a pathogenic bacterial species, but there was little effect on indicators of physiological challenge and other immunological measures.     

REPRODUCTIVE BIOLOGY OF PETAL COLOR VARIANTS IN WILD POPULATIONS OF RAPHANUS SATIVUS: I. POLLINATOR RESPONSE TO COLOR MORPHS

Pollinator response to petal color polymorphism in wild radish (Raphanus sativus) was investigated. Behavior of insect visitors was observed within experimental flower arrays, each containing two of the petal color forms seen intermixed in California populations: white, yellow, pink, and bronze. Honeybees, which accounted for almost 90% of all visits, typically preferred yellow or white flowers and discriminated against bronze. Their preference for white increased significantly as the Raphanus flowering season progressed. Syrphid flies were also frequent visitors and increased in abundance near the end of the season. Syrphids typically preferred pink to other colors. Individual honeybees tended to specialize on either yellow or pink flowers on a short-term basis. This foraging pattern provides the potential for positive assortative mating among plants with yellow or pink flowers. Intraspecific pollinator discrimination may influence genotypic frequencies as well as the relative maternal and paternal reproductive success of color morphs.     

Twin‐arginine translocation system (tat) mutants of Salmonella are attenuated due to envelope defects,not respiratory defects

The twin‐arginine translocation system (Tat) transports folded proteins across the cytoplasmic membrane and is critical to virulence in Salmonella and other pathogens. Experimental and bioinformatic data indicate that 30 proteins are exported via Tat in Salmonella Typhimurium. However, there are no data linking specific Tat substrates with virulence. We inactivated every Tat‐exported protein and determined the virulence phenotype of mutant strains. Although a tat mutant is highly attenuated, no single Tat‐exported substrate accounts for this virulence phenotype. Rather, the attenuation is due primarily to envelope defects caused by failure to translocate three Tat substrates, the N‐acetylmuramoyl‐l ‐alanine amidases, AmiA and AmiC, and the cell division protein, SufI. Strikingly, neither the amiA amiC nor the sufI mutations alone conferred any virulence defect. Although AmiC and SufI have previously been localized to the divisome, the synthetic phenotypes observed are the first to suggest functional overlap. Many Tat substrates are involved in anaerobic respiration, but we show that a mutant completely deficient in anaerobic respiration retains full virulence in both the oral and systemic phases of infection. Similarly, an obligately aerobic mutant is fully virulent. These results suggest that in the classic mouse model of infection, S. Typhimurium is replicating only in aerobic environments.     

Nitrogen Utilization and Toxin Production by Two Diatoms of the Pseudo‐nitzschia pseudodelicatissima Complex: P. cuspidata and P. fryxelliana

The toxigenic diatom Pseudo‐nitzschia cuspidata, isolated from the U.S. Pacific Northwest, was examined in unialgal batch cultures to evaluate domoic acid (DA) toxicity and growth as a function of light, N substrate, and growth phase. Experiments conducted at saturating (120 μmol photons · m^?2 · s^?1) and subsaturating (40 μmol photons · m^?2 · s^?1) photosynthetic photon flux density (PPFD), demonstrate that P. cuspidata grows significantly faster at the higher PPFD on all three N substrates tested [nitrate (NO₃^?), ammonium (NH₄⁺), and urea], but neither cellular toxicity nor exponential growth rates were strongly associated with one N source over the other at high PPFD. However, at the lower PPFD, the exponential growth rates were approximately halved, and the cells were significantly more toxic regardless of N substrate. Urea supported significantly faster growth rates, and cellular toxicity varied as a function of N substrate with NO₃^?‐supported cells being significantly more toxic than both NH₄⁺‐ and urea‐supported cells at the low PPFD. Kinetic uptake parameters were determined for another member of the P. pseudodelicatissima complex, P. fryxelliana. After growth of these cells on NO₃^? they exhibited maximum specific uptake rates (V_max) of 22.7, 29.9, 8.98 × 10^?3 · h^?1, half‐saturation constants (K_s) of 1.34, 2.14, 0.28 μg‐at N · L^?1, and affinity values (α) of 17.0, 14.7, 32.5 × 10^?3 · h^?1/(μg‐at N · L^?1) for NO₃^?, NH₄⁺ and urea, respectively. These labo‐ratory results demonstrate the capability of P. cuspidata to grow and produce DA on both oxidized and reduced N substrates during both exponential and stationary growth phases, and the uptake kinetic results for the pseudo‐cryptic species, P. fryxelliana suggest that reduced N sources from coastal runoff could be important for maintenance of these small pennate diatoms in U.S. west coast blooms, especially during times of low ambient N concentrations.