Whole Proteome Analysis of Mouse Lymph Nodes in Cutaneous Anthrax

This study aimed to characterize a soluble proteome of popliteal lymph nodes during lymphadenitis induced by intradermal injection of Bacillus anthracis Sterne spores in mice using tandem LC-MS/MS and reverse-phase protein microarray with antibodies specific to epitopes of phosphorylated proteins. More than 380 proteins were detected in the normal intra-nodal lymph, while the infectious process resulted in the profound changes in the protein abundances and appearance of 297 unique proteins. These proteins belong to an array of processes reflecting response to wounding, inflammation and perturbations of hemostasis, innate immune response, coagulation and fibrinolysis, regulation of body fluid levels and vascular disturbance among others. Comparison of lymph and serum revealed 83 common proteins. Also, using 71 antibodies specific to total and phosphorylated forms of proteins we carried initial characterization of circulating lymph phosphoproteome which brought additional information regarding signaling pathways operating in the lymphatics. The results demonstrate that the proteome of intra-nodal lymph serves as a sensitive sentinel of the processes occurring within the lymph nodes during infection. The acute innate response of the lymph nodes to anthrax is accompanied by cellular damage and inflammation with a large number of up- and down-regulated proteins many of which are distinct from those detected in serum. MS data are available via ProteomeXchange with identifier PXD001342.     

First report on chitinous holdfast in sponges (Porifera)

A holdfast is a root- or basal plate-like structure of principal importance that anchors aquatic sessile organisms, including sponges, to hard substrates. There is to date little information about the nature and origin of sponges’ holdfasts in both marine and freshwater environments. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within holdfasts of the endemic freshwater demosponge Lubomirskia baicalensis. Using a variety of techniques (near-edge X-ray absorption fine structure, Raman, electrospray ionization mas spectrometry, Morgan–Elson assay and Calcofluor White staining), we show that chitin from the sponge holdfast is much closer to α-chitin than to β-chitin. Most of the three-dimensional fibrous skeleton of this sponge consists of spicule-containing proteinaceous spongin. Intriguingly, the chitinous holdfast is not spongin-based, and is ontogenetically the oldest part of the sponge body. Sequencing revealed the presence of four previously undescribed genes encoding chitin synthases in the L. baicalensis sponge. This discovery of chitin within freshwater sponge holdfasts highlights the novel and specific functions of this biopolymer within these ancient sessile invertebrates.     

The eye of the freshwater prosobranch gastropod Viviparus viviparus: ultrastructure, electrophysiology and behaviour

We used light and electron microscopy to study the retinal organization of the eye of Viviparus viviparus. Electroretinogram (ERG) recordings were used to investigate the electrophysiological responsiveness to flashes of light of varying intensity and colour, behavioural observations were made of phototactic reactions, and optical measurements and calculations related to the path of light rays in the eye were made. The retina contains principally two types of cells: first, photoreceptor cells with both microvilli and cilia, and second, cells, often strongly pigmented, that are supportive in nature. The ERGs obtained were essentially similar in form, amplitude and duration to those known from other gastropods that have exclusively rhabdomeric photoreceptors. Spectral sensitivity curves closely fitted the absorption spectrum of a rhodopsin‐like pigment. The spectral sensitivity peak was at 475 nm. Measurements of the refractive indices of the lens gave values of 1.55 for the outer layer and 1.57 for the lens core. None of the snails tested exhibited a ‘defensive reflex’ and although no preference between light and dark regions was expressed, we nevertheless argue that, on the basis of optical measurements and calculations, the eye of V. viviparus is well‐adapted for seeing under water. Our main conclusion is that in the eye of V. viviparus with its ‘mixed photoreceptor’ cell type, there is an equal probability for microvilli and cilia to function as principal photoreceptive elements.     

Protein Composition Reflects Extracellular Vesicle Heterogeneity

Extracellular vesicles (EVs) are membrane‐enclosed particles that are released by virtually all cells from all living organisms. EVs shuttle biologically active cargo including protein, RNA, and DNA between cells. When shed by cancer cells, they function as potent intercellular messangers with important functional consequences. Cells produce a diverse spectrum of EVs, spanning from small vesicles of 40–150 nm in diameter, to large vesicles up to 10 μm in diameter. While this diversity was initially considered to be purely based on size, it is becoming evident that different classes of EVs, and different populations within one EV class may harbor distinct molecular cargo and play specific functions. Furthermore, there are considerable cell type‐dependent differences in the cargo and function of shed EVs. This review focuses on the most recent proteomic studies that have attempted to capture the EV heterogeneity by directly comparing the protein composition of different EV classes and EV populations derived from the same cell source. Recent studies comparing protein composition of the same EV class(es) derived from different cell types are also summarized. Emerging approaches to study EV heterogeneity and their important implications for future studies are also discussed.     

Construction of simplified models to simulate estrogenic disruptions by esters of 4-hydroxy benzoic acid (parabens)

Four parabens (methyl, n-butyl, benzyl and isobutylparaben) were theoretically studied in order to evaluate their estrogenic activity through simplified models. The experimental structure of the human estrogen receptor ligand-binding domain in complex with 17β-estradiol was used as the starting point to construct the models. The complex between 17β-estradiol and three fragments of the estrogenic receptor (Arg, Glu and His), resulted in a reasonable simplified model of interaction. The replacement of 17-β-estradiol by parabens was evaluated by conformational analyses and interaction energy calculations at BHandHLYP/cc-PVTZ(-f)+ level of theory. According with the calculated interaction energies, methylparaben is the paraben with higher estrogenic activity, which is in agreement with experimental studies of extraction and quantification of parabens in tumors. The antibacterial activity of parabens was also explored considering the formation of potassium salts in the phenolic OH groups. From the obtained relative energy values, methylparaben is the most active preservative.     

Identification and functional analysis of the essential and regulatory light chains of the only type II myosin Myo1p in Saccharomyces cerevisiae

Cytokinesis in Saccharomyces cerevisiae involves coordination between actomyosin ring contraction and septum formation and/or targeted membrane deposition. We show that Mlc1p, a light chain for Myo2p (type V myosin) and Iqg1p (IQGAP), is the essential light chain for Myo1p, the only type II myosin in S. cerevisiae. However, disruption or reduction of Mlc1p-Myo1p interaction by deleting the Mlc1p binding site on Myo1p or by a point mutation in MLC1, mlc1-93, did not cause any obvious defect in cytokinesis. In contrast, a different point mutation, mlc1-11, displayed defects in cytokinesis and in interactions with Myo2p and Iqg1p. These data suggest that the major function of the Mlc1p-Myo1p interaction is not to regulate Myo1p activity but that Mlc1p may interact with Myo1p, Iqg1p, and Myo2p to coordinate actin ring formation and targeted membrane deposition during cytokinesis. We also identify Mlc2p as the regulatory light chain for Myo1p and demonstrate its role in Myo1p ring disassembly, a function likely conserved among eukaryotes.     

Systemic cytokine response in murine anthrax

Systemic pro-inflammatory cytokine release has been previously implicated as a major death-causing factor in anthrax, however, direct data have been absent. We determined the levels of IL-1 beta, IL-6 and TNF-alpha in serum of mice challenged with virulent (Ames) or attenuated (Sterne) strains of Bacillus anthracis. More than 10-fold increase in the IL-1beta levels was detected in Ames-challenged Balb/c mice, in contrast to more susceptible C57BL/6 mice, which showed no IL-1beta response. Balb/c mice have also responded with higher levels of IL-6. The A/J mice demonstrated IL-1beta and IL-6 systemic response to either Ames or Sterne strain of B. anthracis, whereas no increase in TNF-alpha was detected in any murine strain. We used RT-PCR for gene expression analyses in the liver which often is a major source of cytokines and one of the main targets in infectious diseases. A/J mice challenged with B. anthracis (Sterne) showed increased gene expression for Fas, FasL, Bax, IL-1 beta, TNF-alpha, TGF-beta, MIP-1alpha, KC and RANTES. These data favour the hypothesis that apoptotic cell death during anthrax infection causes chemokine-induced transmigration of inflammatory cells to vitally important organs such as liver. Administration of caspase inhibitors z-VAD-fmk and ac-YVAD-cmk improved survival in Sterne-challenged mice indicating a pathogenic role of apoptosis in anthrax.     

Nested species interactions promote feasibility over stability during the assembly of a pollinator community

The foundational concepts behind the persistence of ecological communities have been based on two ecological properties: dynamical stability and feasibility. The former is typically regarded as the capacity of a community to return to an original equilibrium state after a perturbation in species abundances and is usually linked to the strength of interspecific interactions. The latter is the capacity to sustain positive abundances on all its constituent species and is linked to both interspecific interactions and species demographic characteristics. Over the last 40 years, theoretical research in ecology has emphasized the search for conditions leading to the dynamical stability of ecological communities, while the conditions leading to feasibility have been overlooked. However, thus far, we have no evidence of whether species interactions are more conditioned by the community''s need to be stable or feasible. Here, we introduce novel quantitative methods and use empirical data to investigate the consequences of species interactions on the dynamical stability and feasibility of mutualistic communities. First, we demonstrate that the more nested the species interactions in a community are, the lower the mutualistic strength that the community can tolerate without losing dynamical stability. Second, we show that high feasibility in a community can be reached either with high mutualistic strength or with highly nested species interactions. Third, we find that during the assembly process of a seasonal pollinator community located at The Zackenberg Research Station (northeastern Greenland), a high feasibility is reached through the nested species interactions established between newcomer and resident species. Our findings imply that nested mutualistic communities promote feasibility over stability, which may suggest that the former can be key for community persistence.     

Comparative characteristic of Mytilus muscle cells developed in vitro and in vivo

The mussel cells from premyogenic larval stages are capable of differentiation into smooth muscle cells in vitro. However, the behavior and protein composition of these cells are not completely identical to those of smooth muscle cells of adult mussels. In this study we compared some properties of mussel muscle cells forming from cells of trochophore (premyogenic larval stage) in vitro with those of muscle cells of veliger and adult mussel. We found a substantial difference between the contractile apparatus protein composition of veliger muscle and cultivated cells. Myorod, one of the molecular markers of the phenotype of mollusc smooth muscle cells (Shelud'ko et al., 1999, Comp Biochem Physiol 122:277-285), is not a constituent of the contractile apparatus of veliger muscle. At the same time the protein composition of contractile apparatus in cultivated cells was similar to that of adult Mytilus muscles. There were only few quantitative differences between them. The contractile activity of cultivated cells was changing in time. The kinetic parameters of first spontaneous contractions were similar to those of phasic contractions, while their period was close to that of tonic contractions. After 50-55 hrs cultivation the cells produced both phasic and tonic contractions, but the character of contractile activity of cultivated cells was regulated after six days of cultivation only. However, there were no muscle cells in vitro, whose contractile activity was similar to that of veliger muscle cells. So, we concluded that properties of muscle cells forming from premyogenic larval mussel cells in culture are similar to those of muscle cells of the adult mussel, but not of veliger.