Probing the substrate-binding sites of aminoacyl-tRNA synthetases with the procion dye green HE-4BD.

In contrast with previous reports, it was found that membrane-protein phosphorylation by the catalytic subunit (CS) of cyclic AMP-dependent protein kinase had no effect on Ca2+ uptake into platelet membrane vesicles or on subsequent Ca2+ release by inositol 1,4,5-trisphosphate (IP3). Furthermore, IP-20, a highly potent synthetic peptide inhibitor of CS, which totally abolished membrane protein phosphorylation by endogenous or exogenous CS, also had no effect on either Ca2+ uptake or release by IP3. Commercial preparations of protein kinase inhibitor protein (PKI) usually had no effect, but one preparation partially inhibited Ca2+ uptake, which is attributable to the gross impurity of the commercial PKI preparation. IP3-induced release of Ca2+ was also unaffected by the absence of ATP from the medium, supporting the conclusion that Ca2+ release by IP3 does not require the phosphorylation of membrane protein.     

A novel and high-throughput approach to assess photosynthetic thermal tolerance of kelp using chlorophyll α fluorometry

Foundation seaweed species are experiencing widespread declines and localized extinctions due to increased instability of sea surface temperature. Characterizing temperature thresholds are useful for predicting patterns of change and identifying species most vulnerable to extremes. Existing methods for characterizing seaweed thermal tolerance produce diverse metrics and are often time-consuming, making comparisons between species and techniques difficult, hindering insight into global patterns of change. Using three kelp species, we adapted a high-throughput method – previously used in terrestrial plant thermal biology – for use on kelps. This method employs temperature-dependent fluorescence (T–F₀) curves under heating or cooling regimes to determine the critical temperature (T_crit) of photosystem II (PSII), i.e., the breakpoint between slow and fast rise fluorescence response to changing temperature, enabling rapid assays of photosynthetic thermal tolerance using a standardized metric. This method enables characterization of T_crit for up to 48 samples per two-hour assay, demonstrating the capacity of T–F₀ curves for high-throughput assays of thermal tolerance. Temperature-dependent fluorescence curves and their derived metric, T_crit, may offer a timely and powerful new method for the field of phycology, enabling characterization and comparison of photosynthetic thermal tolerance of seaweeds across many populations, species, and biomes.     

Time Savers for Fixing and Dehydration

A double wash bottle for dispensing two-part fixing solutions is described. Equal volumes of each stock solution are delivered simultaneously into the same vial.     

THE EVOLUTION OF LOCOMOTOR RHYTHMICITY IN TETRAPODS

Differences in rhythmicity (relative variance in cycle period) among mammal, fish, and lizard feeding systems have been hypothesized to be associated with differences in their sensorimotor control systems. We tested this hypothesis by examining whether the locomotion of tachymetabolic tetrapods (birds and mammals) is more rhythmic than that of bradymetabolic tetrapods (lizards, alligators, turtles, salamanders). Species averages of intraindividual coefficients of variation in cycle period were compared while controlling for gait and substrate. Variance in locomotor cycle periods is significantly lower in tachymetabolic than in bradymetabolic animals for datasets that include treadmill locomotion, non‐treadmill locomotion, or both. When phylogenetic relationships are taken into account the pooled analyses remain significant, whereas the non‐treadmill and the treadmill analyses become nonsignificant. The co‐occurrence of relatively high rhythmicity in both feeding and locomotor systems of tachymetabolic tetrapods suggests that the anatomical substrate of rhythmicity is in the motor control system, not in the musculoskeletal components.     

Resident mesenchymal cells and fibrosis

Fibrosis is a major clinical problem associated with as many as 45% of all natural deaths in developed nations.It can affect all organs and accumulating evidence indicates that fibrogenesis is not merely a bystander product of injury, but is a central pathological problem directly contributing to loss of organ function. In the majority of clinical cases, fibrogenesis is strongly associated with the recruitment of leukocytes, even in the absence of infection. Although chronic infections are a significant cause of fibrogenesis, in most cases fibrotic disease occurs in the context of sterile injury, such as microvascular disease, toxic epithelial injury or diabetes mellitus. Fibrogenesis is a direct consequence of the activation of extensive, and previously poorly appreciated, populations of mesenchymal cells in our organs which are either wrapped around capillaries and known as ‘pericytes’, or embedded in interstitial spaces between cell structures and known as resident ‘fibroblasts’. Recent fate-mapping and complementary studies in several organs indicate that these cells are the precursors of the scar-forming myofibroblasts that appear in our organs in response to injury. Here we will review the literature supporting a central role for these cells in fibrogenesis, and highlight some of the critical cell to cell interactions that are necessary for the initiation and continuation of the fibrogenic process. This article is part of a Special Issue entitled: Fibrosis: Translation of basic research to human disease.     

Spatial patterns of extra‐pair paternity for spotted towhees Pipilo maculatus in urban parks

The extra‐pair (EP) mating system of birds may be influenced by food resources, such that nutritionally stressed females are unable to pursue EP fertilizations (constrained female hypothesis; CFH), or that females on poor territories acquire EP fertilizations during extra‐territorial forays in search of food (mating opportunity hypothesis; MOH). Edges of urban habitat fragments are sites of apparent high food abundance for spotted towhees Pipilo maculatus, and we used distance to habitat edge in four urban parks in Portland, OR, USA (2004–2006), to test the CFH and MOH. EP paternity was independent of park identity and year; 44% of nests contained EP young and 26% of all young were EP. As predicted by the CFH, EP paternity was more common in nests of long‐tailed (presumably) high quality females. However, independently of tail length, younger females had more EP young than older females, a finding consistent with the MOH. Contrary to predictions of both hypotheses, the probability that a nest contained EP young was highest both at the habitat edge and habitat interior while the proportion of young in nests of EP origin (for nests with EP young) was highest at intermediate distances from habitat edge. We propose that high frequency of EP paternity among females in the interior occurred because, as predicted by the MOH, they ranged more widely in search of food and often encountered EP males. High probability of EP paternity near edges was likely unrelated to female quality. Instead, anthropogenic food sources may have attracted individuals to edges and increased encounters between potential EP mates. Simple opportunity seems likely to account for patterns of EP paternity in spotted towhees, suggesting that human altered environments have the potential to substantially affect EP mating behavior.     

A Foundation for Reliable Spatial Proteomics Data Analysis

Quantitative mass-spectrometry-based spatial proteomics involves elaborate, expensive, and time-consuming experimental procedures, and considerable effort is invested in the generation of such data. Multiple research groups have described a variety of approaches for establishing high-quality proteome-wide datasets. However, data analysis is as critical as data production for reliable and insightful biological interpretation, and no consistent and robust solutions have been offered to the community so far. Here, we introduce the requirements for rigorous spatial proteomics data analysis, as well as the statistical machine learning methodologies needed to address them, including supervised and semi-supervised machine learning, clustering, and novelty detection. We present freely available software solutions that implement innovative state-of-the-art analysis pipelines and illustrate the use of these tools through several case studies involving multiple organisms, experimental designs, mass spectrometry platforms, and quantitation techniques. We also propose sound analysis strategies for identifying dynamic changes in subcellular localization by comparing and contrasting data describing different biological conditions. We conclude by discussing future needs and developments in spatial proteomics data analysis.