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This article describes a patient whose cutaneous pyogenic granuloma was mistaken for infection after injury from a fractured smartphone screen. Clinicians should suspect pyogenic granuloma in patients with these types of injuries so that patients can avoid unnecessary procedures, antibiotics, and discomfort.

P bodies are archetypal biomolecular condensates that concentrate proteins and RNA without a surrounding membrane. While dozens of P body proteins are known, the concentrations of components in the compartment have not been measured. We used live cell imaging to generate a quantitative inventory of the major proteins in yeast P bodies. Only 7 proteins are highly concentrated in P bodies (5.1-15 μM); the 24 others examined are appreciably lower (most ≤ 2.4 μM). P body concentration correlates inversely with cytoplasmic exchange rate. Sequence elements driving Dcp2 concentration into P bodies are distributed across the protein and act cooperatively. Strong correlation between interaction valency of components and P body concentration suggests how the composition of other condensates may be estimated from known interaction data. Our data indicate that P bodies, and probably other condensates, are biochemically simpler than suggested by proteomic analyses, with implications for specificity, reconstitution and evolution.

Histones are located in the cell nucleus. They are positively charged small proteins which became extracellular upon apoptosis, necrosis, and infection – induced cell death. The mixture of extracellular Histones was shown to bundle Actin filaments and digested by bacterial proteases, which was inhibited by DNA and Actin. Here we studied the interaction of five major family of Histones, H2A, H2B, H3.1, H1 and H4, with DNA and Actin filaments. We found that all the Histones studied bound to DNA, increased the viscosity of Actin containing solutions and bundled Actin filaments in various degrees. The bundling of Actin filaments by Histones was inhibited by DNA, NaCl and DNase1. DNA and Actin filaments also inhibited the proteolysis of the five Histones by Subtilisin, Fusolisin and Pseudomonas Aeruginosa bacterial proteases. Both the degree of the proteolysis and its inhibition was different with various Histones. The results indicate that all the Histones studied bind strongly to the negatively charged DNA and to the Actin filaments.

PARN loss-of-function mutations cause a severe form of the hereditary disease dyskeratosis congenita (DC). PARN deficiency affects the stability of non-coding RNAs such as human telomerase RNA (hTR), but these effects do not explain the severe disease in patients. We demonstrate that PARN deficiency affects the levels of numerous miRNAs in human cells. PARN regulates miRNA levels by stabilizing either mature or precursor miRNAs by removing oligo(A) tails added by the poly(A) polymerase PAPD5, which if remaining recruit the exonuclease DIS3L or DIS3L2 to degrade the miRNA. PARN knockdown destabilizes multiple miRNAs that repress p53 translation, which leads to an increase in p53 accumulation in a Dicer-dependent manner, thus explaining why PARN-defective patients show p53 accumulation. This work also reveals that DIS3L and DIS3L2 are critical 3' to 5' exonucleases that regulate miRNA stability, with the addition and removal of 3' end extensions controlling miRNA levels in the cell.

Stress granules and P-bodies are conserved assemblies of nontranslating mRNAs in eukaryotic cells that can be related to RNA–protein aggregates found in some neurodegenerative diseases. Herein, we examine how the Hsp70/Hsp40 protein chaperones affected the assembly and disassembly of stress granules and P-bodies in yeast. We observed that Hsp70 and the Ydj1 and Sis1 Hsp40 proteins accumulated in stress granules and defects in these proteins led to decreases in the disassembly and/or clearance of stress granules. We observed that individual Hsp40 proteins have different effects on stress granules with defects in Ydj1 leading to accumulation of stress granules in the vacuole and limited recovery of translation following stress, which suggests that Ydj1 promotes disassembly of stress granules to promote translation. In contrast, defects in Sis1 did not affect recovery of translation, accumulated cytoplasmic stress granules, and showed reductions in the targeting of stress granules to the vacuole. This demonstrates a new principle whereby alternative disassembly machineries lead to different fates of components within stress granules, thereby providing additional avenues for regulation of their assembly, composition, and function. Moreover, a role for Hsp70 and Hsp40 proteins in stress granule disassembly couples the assembly of these stress responsive structures to the proteostatic state of the cell.

Host defense peptides are effector molecules of the innate immunity that possess antimicrobial and health-promoting properties. Due to their potential therapeutic activities, host defense peptides are being explored as alternatives for antibiotics. The human LL-37 and its shorter, cost-effective, bactericidal core peptide derivates have been suggested for their therapeutic potential. Bacteria evade host defense peptides by proteolytic inactivation. Actin released from necrotized cells and abundant in infected sites was shown to bind and protect LL-37 from microbial proteolytic degradation, and to enable the peptide’s antimicrobial action despite the presence of the proteases. Here, we characterized the interactions of the 10–13 residues long LL-37 core peptides with actin. We show that the LL-37 core peptides associate with actin with a lower affinity than that of LL-37. Their association with actin, which is very ionic strength sensitive, is mainly based on electrostatic interactions. Likewise, the antimicrobial activity against Escherichia coli of the minimal antimicrobial peptide KR-12 but not FK-13 nor LL-37 is also very sensitive to salts. In addition, the antimicrobial activity of the FK-13 core peptide is protected by actin against the tested bacterial proteases in a similar manner to that of LL-37, supporting its potential for therapeutic use.

Histones are small polycationic proteins complexed with DNA located in the cell nucleus. Upon apoptosis they are secreted from the cells and react with extracellular polyanionic compounds. Actin which is a polyanionic protein, is also secreted from necrotic cells and interacts with histones. We showed that both histone mixture (histone type III) and the recombinant H2A histone bundles F-actin, increases the viscosity of the F-actin containing solution and polymerizes G-actin. The histone-actin bundles are relatively insensitive to increase of ionic strength, unlike other polycation, histatin, lysozyme, spermine and LL-37 induced F-actin bundles. The histone-actin bundles dissociate completely only in the presence of 300-400 mM NaCl. DNA, which competes with F-actin for histones, disassembles histone induced actin bundles. DNase1, which depolymerizes F- to G-actin, actively unbundles the H2A histone induced but slightly affects the histone mixture induced actin bundles. Cofilin decreases the amount of F-actin sedimented by low speed centrifugation, increases light scattering and viscosity of F-actin-histone mixture containing solutions and forms star like superstructures by copolymerizing G-actin with H2A histone. The results indicate that histones are tightly attached to F-actin by strong electrostatic and hydrophobic forces. Since both histones and F-actin are present in the sputum of patients with cystic fibrosis, therefore, the formation of the stable histone-actin bundles can contribute to the pathology of this disease by increasing the viscosity of the sputum. The actin-histone interaction in the nucleus might affect gene expression.

The evolutionarily conserved Lsm1-7-Pat1 complex is the most critical activator of mRNA decapping in eukaryotic cells and plays many roles in normal decay, AU-rich element-mediated decay, and miRNA silencing, yet how Pat1 interacts with the Lsm1-7 complex is unknown. Here, we show that Lsm2 and Lsm3 bridge the interaction between the C-terminus of Pat1 (Pat1C) and the Lsm1-7 complex. The Lsm2-3-Pat1C complex and the Lsm1-7-Pat1C complex stimulate decapping in vitro to a similar extent and exhibit similar RNA-binding preference. The crystal structure of the Lsm2-3-Pat1C complex shows that Pat1C binds to Lsm2-3 to form an asymmetric complex with three Pat1C molecules surrounding a heptameric ring formed by Lsm2-3. Structure-based mutagenesis revealed the importance of Lsm2-3-Pat1C interactions in decapping activation in vivo. Based on the structure of Lsm2-3-Pat1C, a model of Lsm1-7-Pat1 complex is constructed and how RNA binds to this complex is discussed.

Purpose To explore the efficacy of injectable collagenase Clostridium histolyticum (CCH) in distal interphalangeal (DIP) joint contractures in Dupuytren disease . Methods A retrospective review was conducted for patients with DIP joint contractures treated with CCH. Baseline contracture and posttreatment residual contracture were recorded. The primary end point was achievement of 0° to 5° of residual contracture 30 days postmanipulation. Recurrence and adverse events are also reported. Results Twenty-one patients were treated with collagenase for DIP joint contractures and had a mean follow-up of 2.6 months. Seventeen of 21 patients reached the primary end point of less than 5° residual flexion contracture . Eleven of 21 patients experienced an adverse event, most commonly bruising and edema. No serious complications such as neurovascular injuries or tendon ruptures were recorded. Two patients had recurrence of disease. Conclusions Injection with CCH is an option for the treatment of DIP joint contractures in Dupuytren disease. Type of study/level of evidence Therapeutic IV.

Histones are small polycationic proteins located in the cell nucleus. Together, DNA and histones are integral constituents of the nucleosomes. Upon apoptosis, necrosis, and infection – induced cell death, histones are released from the cell. The extracellular histones have strong antimicrobial activity but are also cytotoxic and thought as mediators of cell death in sepsis. The antimicrobial activity of the cationic extracellular histones is inhibited by the polyanionic DNA and F-actin, which also become extracellular upon cell death. DNA and F-actin protect histones from degradation by the proteases of Pseudomonas aeruginosa and Porphyromonas gingivalis. However, though the integrity of the histones is protected, the activity of histones as antibacterial agents is lost. The inhibition of the histone’s antibacterial activity and their protection from proteolysis by DNA and F-actin indicate a tight electrostatic interaction between the positively charged histones and negatively charged DNA and F-actin, which may have physiological significance in maintaining the equilibrium between the beneficial antimicrobial activity of extracellular histones and their cytotoxic effects.

The objective of this paper is the presentation of the European Road Safety Knowledge System, which was developed within the DaCoTA research project of the 7th Framework Programme of the European Commission. This knowledge system includes a number of components, concerning data and tools, road safety issues and countries. A wealth of data was gathered, on road fatalities, exposure, safety performance indicators, socioeconomic indicators, health and causation indicators, road user attitudes & behaviours, traffic laws and regulations, road safety management etc., for 30 European countries. This data was used in road safety analyses leading to the Basic Fact Sheets, and the Annual Statistical report. Comprehensive syntheses of the literature on important road safety issues and methodologies were carried out, in the form of web-texts. Country analyses were also carried out, including road safety management “profiles”, “country overviews” and country forecasts. These were integrated through a web-based Road Safety Knowledge System. This System can be a very useful support for road safety research in Europe, and ideal to link research and policy making.

The objective of this paper is the analysis of road safety management in European countries and the identification of “good practice”. A road safety management investigation model was created, based on several “good practice” criteria. Road safety management systems have been thoroughly investigated in 14 European countries on 2010, by means of interviews with both governmental representatives and independent experts, who filled in an extensive questionnaire. A reliable and accurate picture (“profile”) was created for each country, allowing country comparisons. Then, statistical methods were used to make rankings of countries, and analyse the relationship between road safety management and road safety performance. The results of the analyses suggest that it is not possible to identify one single “good practice”. Nevertheless, there were several elements that emerged as “good practice” criteria. On the basis of the results, recommendations are proposed at national and European level.

The objective of this research is the assessment of current needs for evidence-based road safety decision making in Europe, through the consultation of a panel of road safety experts. The members of this Experts Panel have extensive knowledge of road safety management processes and needs in their country, being either directly involved in decision making, or working closely with decision makers. Two consultation methods were implemented: semi-directive interviews and written contributions. The synthesis of the results was carried out by means of a predefined matrix, in which the road safety management tasks were separated into their components, and were then cross-tabulated with distinct categories of needs. The results provide valuable information on the current and future needs for evidence-based road safety management in Europe. A number of key issues were brought forward with wide consensus among Experts, such as the need to make the consideration of scientific evidence in road safety decisions compulsory in all countries. The establishment of appropriate procedures was emphasized, including institutional arrangements for road safety management, with the necessary links and interactive procedures for local needs. The results also include useful recommendations, for the setting of targets, the use of cost-benefit analyses, the analysis of combined effects of measures, the collection of data on measures implementation, the collection of exposure and behavioural data, the estimation of injury under-reporting and the standardization of analysis methods.

Three polycations, polylysine, the polyamine spermine and the polycationic protein lysozyme were used to study the formation, structure, ionic strength sensitivity and dissociation of polycation-induced actin bundles. Bundles form fast, simultaneously with the polymerization of MgATP-G-actins, upon the addition of polycations to solutions of actins at low ionic strength conditions. This indicates that nuclei and/or nascent filaments bundle due to attractive, electrostatic effect of polycations and the neutralization of repulsive interactions of negative charges on actin. The attractive forces between the filaments are strong, as shown by the low (in nanomolar range) critical concentration of their bundling at low ionic strength. These bundles are sensitive to ionic strength and disassemble partially in 100 mM NaCl, but both the dissociation and ionic strength sensitivity can be countered by higher polycation concentrations. Cys374 residues of actin monomers residing on neighboring filaments in the bundles can be cross-linked by the short span (5.4 A) MTS-1 (1,1-Methanedyl Bismethanethiosulfonate) cross-linker, which indicates a tight packing of filaments in the bundles. The interfilament cross-links, which connect monomers located on oppositely oriented filaments, prevent disassembly of bundles at high ionic strength. Cofilin and the polysaccharide polyanion heparin disassemble lysozyme induced actin bundles more effectively than the polylysine-induced bundles. The actin-lysozyme bundles are pathologically significant as both proteins are found in the pulmonary airways of cystic fibrosis patients. Their bundles contribute to the formation of viscous mucus, which is the main cause of breathing difficulties and eventual death in this disorder.

Cap hydrolysis is a critical control point in the life of eukaryotic mRNAs and is catalyzed by the evolutionarily conserved Dcp1-Dcp2 complex. In Saccharomyces cerevisiae, decapping is modulated by several factors, including the Lsm family protein Edc3, which directly binds to Dcp2. We show that Edc3 binding to Dcp2 is mediated by a short peptide sequence located C terminal to the catalytic domain of Dcp2. This sequence is required for Edc3 to stimulate decapping activity of Dcp2 in vitro, for Dcp2 to efficiently accumulate in P-bodies, and for efficient degradation of the RPS28B mRNA, whose decay is enhanced by Edc3. In contrast, degradation of YRA1 pre-mRNA, another Edc3-regulated transcript, occurs independently from this region, suggesting that the effect of Edc3 on YRA1 is independent of its interaction with Dcp2. Deletion of the sequence also results in a subtle but significant defect in turnover of the MFA2pG reporter transcript, which is not affected by deletion of EDC3, suggesting that the region affects some other aspect of Dcp2 function in addition to binding Edc3. These results raise a model for Dcp2 recruitment to specific mRNAs where regions outside the catalytic core promote the formation of different complexes involved in mRNA decapping.

Buforin is a cationic antimicrobial peptide (AMP) from the stomach of toads. Buforin II is a derivative of this naturally occurring peptide. Buforin IIB is a synthetic analog of buforin II containing a model α-helical sequence (3xRLLR) at the C-terminus. To further increase the antimicrobial activity and decrease toxicity to eukaryotic cells, new derivatives (buforin III analogs) were designed by substituting amino acids in the buforin IIB sequence. In this work, the antimicrobial activity and the actin- and DNA-binding characteristics of buforin IIIB (RVVRQWPIGRVVRRVVRRVVR) and the newly synthetized buforin IIIE (RLLLRQWPIGRLLRRLLRRLLR) were studied. The antimicrobial activity of buforin IIIB (measured against E. coli and E. faecalis ) was significantly greater than that of buforin IIIE, while both peptides were nontoxic to macrophages at the minimal concentrations required to inhibit microbial growth. Actin, which inhibited the antimicrobial activity of the two buforin III analogs, was bundled by both peptides; however, less buforin IIIE than buforin IIIB was needed for bundling. Higher levels of NaCl were needed to unbundle actin bundled by buforin IIIE than actin bundled by buforin IIIB, which indicates that buforin IIIE binds more strongly to actin than buforin IIIB. Actin bundled by either peptide was dissociated with the same concentration of DNA; however, buforin IIIE bound more strongly to DNA than buforin IIIB. These results contribute to the understanding of the antimicrobial mechanism of cationic AMPs in general and histone-derived peptides in particular.

Recent results indicate that nontranslating mRNAs in eukaryotic cells exist in distinct biochemical states that accumulate in P bodies and stress granules, although the nature of interactions between these particles is unknown. We demonstrate in Saccharomyces cerevisiae that RNA granules with similar protein composition and assembly mechanisms as mammalian stress granules form during glucose deprivation. Stress granule assembly is dependent on P-body formation, whereas P-body assembly is independent of stress granule formation. This suggests that stress granules primarily form from mRNPs in preexisting P bodies, which is also supported by the kinetics of P-body and stress granule formation both in yeast and mammalian cells. These observations argue that P bodies are important sites for decisions of mRNA fate and that stress granules, at least in yeast, primarily represent pools of mRNAs stalled in the process of reentry into translation from P bodies.

Summary A critical step in mRNA degradation is the removal of the 5′ cap structure, which is catalyzed by the Dcp1-Dcp2 complex. The crystal structure of an S . pombe Dcp1p-Dcp2n complex combined with small-angle X-ray scattering analysis (SAXS) reveals that Dcp2p exists in open and closed conformations, with the closed complex being, or closely resembling, the catalytically more active form. This suggests that a conformational change between these open and closed complexes might control decapping. A bipartite RNA-binding channel containing the catalytic site and Box B motif is identified with a bound ATP located in the catalytic pocket in the closed complex, suggesting possible interactions that facilitate substrate binding. Dcp1 stimulates the activity of Dcp2 by promoting and/or stabilizing the closed complex. Notably, the interface of Dcp1 and Dcp2 is not fully conserved, explaining why the Dcp1-Dcp2 interaction in higher eukaryotes requires an additional factor.

Cofilin, a member of the actin-depolymerizing factor (ADF)/cofilin family of proteins, is a key regulator of actin dynamics. Cofilin binds to monomer (G-) and filamentous (F-) actin, severs the filaments, and increases their turnover rate. Electron microscopy studies suggested cofilin interactions with subdomains 2 and 1/3 on adjacent actin protomers in F-actin. To probe for the presence of a cryptic cofilin binding site in subdomain 2 in G-actin, we used transglutaminase-mediated cross-linking, which targets Gln41 in subdomain 2. The cross-linking proceeded with up to 85% efficiency with skeletal alpha-actin and WT yeast actin, yielding a single product corresponding to a 1:1 actin-cofilin complex but was strongly inhibited in Q41C yeast actin (in which Q41 was substituted with cysteine). LC-MS/MS analysis of the proteolytic fragments of this complex mapped the cross-linking to Gln41 on actin and Gly1 on recombinant yeast cofilin. The actin-cofilin (AC) heterodimer was purified on FPLC for analytical ultracentrifugation and electron microscopy analysis. Sedimentation equilibrium and velocity runs revealed oligomers of AC in G-actin buffer. In the presence of excess cofilin, the covalent AC heterodimer bound a second cofilin, forming a 2:1 cofilin/actin complex, as revealed by sedimentation results. Under polymerizing conditions the cross-linked AC formed mostly short filaments, which according to image reconstruction were similar to uncross-linked actin-cofilin filaments. Although a majority of the cross-linking occurs at Gln41, a small fraction of the AC cross-linked complex forms in the Q41C yeast actin mutant. This secondary cross-linking site was sequenced by MALDI-MS/MS as linking Gln360 in actin to Lys98 on cofilin. Overall, these results demonstrate that the region around Gln41 (subdomain 2) is involved in a weak binding of cofilin to G-actin.