A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits

Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.     

Analysis of proximal tubule salt and water transport in standing droplets.

A mathematical model has been developed describing solute and water movement in the renal proximal tubule standing droplet experiment. The model explicitly incorporates the constraint of isosmotic reabsorption. Solute asymmetry due to the unequal distribution of protein, bicarbonate and other solutes between plasma and the standing droplet is shown to be one of the major reabsorptive forces; however, the introduction of an additional reabsorptive mechanism into the system equations is required in order to obtain a quantitative fit with experimental observations. The model demonstrates that limiting concentration gradients can be obtained in the absence of active transport and that their magnitudes will vary inversely with the permeability of the poorly permeant solute. Conversely, situations can occur where active transport will not elicit a limiting gradient. Consequently, previous interpretations of the meaning of limiting gradients and their magnitudes need to be reconsidered. The model further predicts that the technique for measuring non-electrolyte permeability using standing droplet experiments is likely to underestimate the true permeability. Finally, it is shown that a previous theoretical model of standing droplets, which does not explicitly include the constraint of isomotic reabsorption, cannot fit experimental data from proximal tubules.     

TLR2 Mediates Recognition of Live Staphylococcus epidermidis and Clearance of Bacteremia

Background

Staphylococcus epidermidis (SE) is a nosocomial pathogen that causes catheter-associated bacteremia in the immunocompromised, including those at the extremes of age, motivating study of host clearance mechanisms. SE-derived soluble components engage TLR2; but additional signaling pathways have also been implicated, and TLR2 can play complex, at times detrimental, roles in host defense against other Staphylococcal spp. The role of TLR2 in responses of primary blood leukocytes to live SE and in clearance of SE bacteremia, the most common clinical manifestation of SE infection, is unknown.

Methodology/Principal Findings

We studied TLR2-mediated recognition of live clinical SE strain 1457 employing TLR2-transfected cells, neutralizing anti-TLR antibodies and TLR2-deficient mice. TLR2 mediated SE-induced cytokine production in human embryonic kidney cells, human whole blood and murine primary macrophages, in part via recognition of a soluble TLR2 agonist. After i.v. challenge with SE, early (1 h) cytokine/chemokine production and subsequent clearance of bacteremia (24–48 h) were markedly impaired in TLR2-deficient mice.

Conclusions/Significance

TLR2 mediates recognition of live SE and clearance of SE bacteremia in vivo.     

Most nuclear systemic autoantigens are extremely disordered proteins: implications for the etiology of systemic autoimmunity

Patients with systemic autoimmune diseases usually produce high levels of antibodies to self-antigens (autoantigens). The repertoire of common autoantigens is remarkably limited, yet no readily understandable shared thread links these apparently diverse proteins. Using computer prediction algorithms, we have found that most nuclear systemic autoantigens are predicted to contain long regions of extreme structural disorder. Such disordered regions would generally make poor B cell epitopes and are predicted to be under-represented as potential T cell epitopes. Consideration of the potential role of protein disorder may give novel insights into the possible role of molecular mimicry in the pathogenesis of autoimmunity. The recognition of extreme autoantigen protein disorder has led us to an explicit model of epitope spreading that explains many of the paradoxical aspects of autoimmunity – in particular, the difficulty in identifying autoantigen-specific helper T cells that might collaborate with the B cells activated in systemic autoimmunity. The model also explains the experimentally observed breakdown of major histocompatibility complex (MHC) class specificity in peptides associated with the MHC II proteins of activated autoimmune B cells, and sheds light on the selection of particular T cell epitopes in autoimmunity. Finally, the model helps to rationalize the relative rarity of clinically significant autoimmunity despite the prevalence of low specificity/low avidity autoantibodies in normal individuals.     

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo^1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin^3-5 or lysed by freeze/thaw cycles^6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.^1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors^18,19 and a current lack of standardized protocols for platelet preparation.^15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.Download video file.^{(100M, mp4)}     

The Formation of Intracellular Crystals in Midgut Glands of Limnoria lignorum

Certain morphological features of intracellular crystal formation within the midgut glands of Limnoria lignorum (Rathke) have been studied with the electron microscope and cytochemical methods. A correlation has been established between Golgi membranes and formation of the crystals. The Prussian blue reaction reveals quantities of iron localized in the intracellular crystals and in small granular structures seen in the apical region of the cells. These granules can be identified as accumulations of Golgi membranes, with which iron-containing particles are associated. When these membrane configurations are studied with the electron microscope, they can be classified and arranged in an assumed sequence which is thought to represent successive stages in the development of crystals. As the membrane systems become progressively specialized, increasing accumulations of dense granular material appear within their interstices. This material is rich in iron and probably represents the component responsible for the positive Prussian blue reaction. This material also appears to be a precursor substance for iron-containing protein molecules which are synthesized and arranged to make up the crystals. These iron-containing molecules are first deposited in orderly array as double rows of dense particles on certain internal membranes of the specialized Golgi complexes. The membranes later disappear and the particles form definitive crystals by rearrangement into a hexagonal close-packed pattern.