The Response of Haloferax volcanii to Salt and Temperature Stress: A Proteome Study by Label‐Free Mass Spectrometry

In‐depth proteome analysis of the haloarchaeal model organism Haloferax volcanii has been performed under standard, low/high salt, and low/high temperature conditions using label‐free mass spectrometry. Qualitative analysis of protein identification data from high‐pH/reversed‐phase fractionated samples indicates 61.1% proteome coverage (2509 proteins), which is close to the maximum recorded values in archaea. Identified proteins match to the predicted proteome in their physicochemical properties, with only a small bias against low‐molecular‐weight and membrane‐associated proteins. Cells grown under low and high salt stress as well as low and high temperature stress are quantitatively compared to standard cultures by sequential window acquisition of all theoretical mass spectra (SWATH‐MS). A total of 2244 proteins, or 54.7% of the predicted proteome, are quantified across all conditions at high reproducibility, which allowed for global analysis of protein expression changes under these stresses. Of these, 2034 are significantly regulated under at least one stress condition. KEGG pathway enrichment analysis shows that several major cellular pathways are part of H. volcanii’s universal stress response. In addition, specific pathways (purine, cobalamin, and tryptophan) are affected by temperature stress. The most strongly downregulated proteins under all stress conditions, zinc finger protein HVO_2753 and ribosomal protein S14, are found oppositely regulated to their immediate genetic neighbors from the same operon.     

The integrative effects of behavior and morphology on amphibian movement

Animal movement and dispersal are key factors in population dynamics and support complex ecosystem processes like cross‐boundary subsidies. Juvenile dispersal is an important mechanism for many species and often involves navigation in unfamiliar habitats. For species that metamorphose, such as amphibians, this transition from aquatic to terrestrial environments involves the growth and use of new morphological traits (e.g., legs). These traits strongly impact the fundamental ability of an organism to move in novel landscapes, but innate behaviors can regulate choices that result in the realized movements expressed. By assessing the integrative role of morphology and behavior, we can improve our understanding of juvenile movement, particularly in understudied organisms like amphibians. We assessed the roles of morphological (snout‐vent length and relative leg length) and performance (maximal jump distance) traits in shaping the free movement paths, measured through fluorescent powder tracking, in three anuran species, Pacific treefrog (Hyliola regilla), Western toad (Anaxyrus boreas), and Cascades frog (Rana cascadae). We standardized the measurement of these traits to compare the relative role of species' innate differences versus physical traits in shaping movement. Innate differences, captured by species identity, were the most significant factor influencing movement paths via total movement distance and path sinuosity. Relative leg length was an important contributor but significantly interacted with species identity. Maximal jump performance, which was significantly predicted by morphological traits, was not an important factor in movement behavior relative to species identity. The importance of species identity and associated behavioral differences in realized movement provide evidence for inherent species differences being central to the dispersal and movement of these species. This behavior may stem from niche partitioning of these sympatric species, yet it also calls into question assumptions generalizing anuran movement behavior. These species‐level effects are important in framing differences as past research is applied in management planning.     

Level of Toll-like receptor agonist exposure differentially determines chemokine production in humans

Toll-like receptor (TLR) agonists, ubiquitously present in the environment, are key players in activating synthesis of cytokines and chemokines that control normal and pathophysiological processes, including multiple inflammatory diseases. TLR2 and TLR4 respond to bacterial cell wall products. We examined the impact of TLR activation on human immune capacity using stimuli ranging from the low levels seen in most environments to the high concentrations widely used for in vitro studies. Peripheral blood mononuclear cells from 117 healthy children were activated with lipopolysaccharide (TLR4 ligand) or peptidoglycan (TLR2 ligand) over a million-fold range of concentrations. Resulting interleukin-6, CCL2, and CCL22 production were quantified by ELISA. The intensity of cytokine production elicited was linearly related to the intensity of the stimulus up to maximal responses. In marked contrast, chemokine production was not linearly related to agonist concentration. Responses rose with increasing stimulation, and then were markedly reduced (40%-100%, p < 0.0001) in response to the high levels of TLR stimulation most commonly cited. Thus, the levels of TLR4 and TLR2 agonists typically used for in vitro interrogation of immune capacity yield results clearly distinct from those obtained using commonly occurring environmental levels of TLR ligands. These findings demonstrate the importance of utilizing TLR ligands at concentrations more closely mimicking environmental levels when assessing immune capacity.     

CD36 signals to the actin cytoskeleton and regulates microglial migration via a p130Cas complex

The pattern recognition receptor CD36 initiates a signaling cascade that promotes microglial activation and recruitment to beta-amyloid deposits in the brain. In the present study we identify the focal adhesion-associated proteins p130Cas, Pyk2, and paxillin as novel members of the tyrosine kinase signaling pathway downstream of CD36 and show that assembly of this complex is essential for microglial migration. In primary microglia and macrophages exposed to beta-amyloid, the scaffolding protein p130Cas is rapidly tyrosine-phosphorylated and co-localizes with CD36 to membrane ruffles contemporaneous with F-actin polymerization. These beta-amyloid-stimulated events are not detected in CD36 null cells and are dependent on CD36 activation of Src family tyrosine kinases. Fyn, a Src kinase known to interact with CD36, co-precipitates with p130Cas and is an essential upstream intermediate in the signaling pathways leading to phosphorylation of the p130Cas substrate domain. Furthermore, the p130Cas-interacting kinase Pyk2 and the cytoskeletal adapter protein paxillin also demonstrate CD36-dependent phosphorylation, identifying these focal adhesion molecules as additional members of this beta-amyloid signaling cascade. Disruption of this p130Cas complex by small interfering RNA silencing inhibits p44/42 mitogen-activated protein kinase phosphorylation and microglial migration, illustrating the importance of this pathway in microglial activation and recruitment. Together, these data are the first to identify the signaling cascade that directly links CD36 to the actin cytoskeleton and, thus, implicates it in diverse processes such as cellular migration, adhesion, and phagocytosis.     

The Hemolymph of the ascidian Styela plicata (Chordata-Tunicata) contains heparin inside basophil-like cells and a unique sulfated galactoglucan in the plasma

The hemolymph of ascidians (Chordata-Tunicata) contains different types of hemocytes embedded in a liquid plasma. In the present study, heparin and a sulfated heteropolysaccharide were purified from the hemolymph of the ascidian Styela plicata. The heteropolysaccharide occurs free in the plasma, is composed of glucose ( approximately 60%) and galactose ( approximately 40%), and is highly sulfated. Heparin, on the other hand, occurs in the hemocytes, and high performance liquid chromatography of the products formed by degradation with specific lyases revealed that it is composed mainly by the disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4)) (39.7%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(6SO(4)) (38.2%). Small amounts of the 3-O-sulfated disaccharides DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4)) (9.8%) and DeltaUA(2SO(4))-1-->4-beta-d-GlcN(SO(4))(3SO(4))(6SO(4)) (3.8%) were also detected. These 3-O-sulfated disaccharides were demonstrated to be essential for the binding of the hemocyte heparin to antithrombin III. Electron microscopy techniques were used to characterize the ultrastructure of the hemocytes and to localize heparin and histamine in these cells. At least five cell types were recognized and classified as univacuolated and multivacuolated cells, amebocytes, hemoblasts, and granulocytes. Immunocytochemistry showed that heparin and histamine co-localize in intracellular granules of only one type of hemocyte, the granulocyte. These results show for the first time that in ascidians, a sulfated galactoglucan circulates free in the plasma, and heparin occurs as an intracellular product of a circulating basophil-like cell.     

Gene expression dynamics in deer antler: mesenchymal differentiation toward chondrogenesis

Annual re-growth of deer antler represents a unique example of complete organ regeneration. Because antler mesenchymal cells retain their embryonic capacity to develop into cartilage or bone, studying antler development provides a natural system to follow gene expression changes during mesenchymal differentiation toward chondrogenic/osteogenic lineage. To identify novel genes involved either in early events of mesenchymal cell specialization or in robust bone development, we have introduced a 3 K heterologous microarray set-up (deer cDNA versus mouse template). Fifteen genes were differentially expressed; genes for housekeeping, regulatory functions (components of different signaling pathways, including FGF, TGFβ, Wnt), and genes encoding members of the Polycomb group were represented. Expression dynamics for genes are visualized by an expression logo. The expression profile of the gene C21orf70 of unknown function is described along with the effects when over-expressed; furthermore the nuclear localization of the cognate protein is shown. In this report, we demonstrate the particular advantage of the velvet antler model in bone research for: (1) identification of mesenchymal and precartilaginous genes and (2) targeting genes upregulated in robust cartilage development. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.