HIV Activates the Tyrosine Kinase Hck to Secrete ADAM Protease-Containing Extracellular Vesicles

HIV-Nef activates the myeloid cell-typical tyrosine kinase Hck, but its molecular role in the viral life cycle is not entirely understood. We found that HIV plasma extracellular vesicles (HIV pEV) containing/10 proteases and Nef also harbor Hck, and analyzed its role in the context of HIV pEV secretion. Myeloid cells required Hck for the vesicle-associated release of ADAM17. This could be induced by the introduction of Nef and implied that HIV targeted Hck for vesicle-associated ADAM17 secretion from a myeloid compartment. The other contents of HIV-pEV, however, including miRNA and effector protein profiles, as well as the presence of haptoglobin suggested hepatocytes as a possible cellular source. HIV liver tissue analysis supported this assumption, revealing induction of Hck translation, evidence for ADAMprotease activation and HIV infection. Our findings suggest that HIV targets Hck to induce pro-inflammatory vesicles release and identifies hepatocytes as a possible host cell compartment. (c) 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license.Peer reviewe

The microstructure and technological properties of ultra high strength 1100MPa grade strip steel

The article describes the microstructure and the technological properties of a direct quenched ultrahighstrength strip steel with the minimum specific yield strength of 1100MPa. The microstructure of thislow carbon, Mn-Cr-Mo-Cu-Ni alloyed steel consists mainly of auto-tempered lath martensite. Due to thesophisticated thermo-mechanical controlled processing schedule, the martensite transformation takesplace from a fine and uniform austenite grain structure. State-of-the-art steelmaking and continuous castingoperations guarantee a good inclusion cleanness and low level of segregation. The steel has excellent impactand fracture toughness properties with respect to its ultra-high strength level. The determined transitiontemperature for 28J in Charpy-V test and fracture toughness characteristic temperature, T0, were below-100°C. The weldability tests indicated that the impact toughness of the heat affected zone (HAZ) is excellentand there is no significant softening in the HAZ or in the welded joint in the wide range of t8/5 cooling times.The steel allows crack-free bending with a minimum inside bending radius equal to 3 times material thicknessirrespective of the bending direction. In addition, the steel has a good resistance to atmospheric corrosion

Observations of ozone depletion events in a Finnish boreal forest

We investigated the concentrations and vertical profiles of ozone over a 20-year period (1996–2016) at the SMEAR II station in southern Finland. Our results showed that the typical daily median ozone concentrations were in the range of 20–50 ppb with clear diurnal and annual patterns. In general, the profile of ozone concentrations illustrated an increase as a function of heights. The main aim of our study was to address the frequency and strength of ozone depletion events at this boreal forest site. We observed more than a thousand of 10 min periods at 4.2 m, with ozone concentrations below 10 ppb, and a few tens of cases with ozone concentrations below 2 ppb. Among these observations, a number of ozone depletion events that lasted for more than 3 h were identified, and they occurred mainly in autumn and winter months. The low ozone concentrations were likely related to the formation of a low mixing layer under the conditions of low temperatures, low wind speeds, high relative humidities and limited intensity of solar radiation.Peer reviewe

Cut-off values to evaluate exercise-induced asthma in eucapnic voluntary hyperventilation test for children

Background and Aim The eucapnic voluntary hyperventilation (EVH) testing is a diagnostic tool for diagnostics of exercise-induced bronchoconstriction; while the testing has become more common among children, data on the test's feasibility among children remain limited. Our aim was to investigate EVH testing feasibility among children, diagnostic testing cut-off values, and which factors affect testing outcomes. Methods We recruited 134 patients aged 10-16 years with a history of exercise-induced dyspnoea and 100 healthy control children to undergo 6-min EVH testing. Testing feasibility was assessed by the children's ability to achieve >= 70% of the target minute ventilation of 30 times forced expiratory volume in 1 s (FEV1). Bronchoconstriction was assessed as a minimum of 8%, 10%, 12%, 15% or 20% fall in FEV1. Patient characteristics were correlated with EVH outcomes. Results Overall, 98% of the children reached >= 70%, 88% reached >= 80%, 79% reached >= 90% and 62% reached >= 100% of target ventilation in EVH testing; of children with a history of exercise-induced dyspnoea, the decline percentages were as follows: 24% (>= 8% fall), 17% (>= 10% fall), 10% (>= 12% fall), 6% (>= 15% fall) and 5% (>= 20% fall) in FEV1, compared to 11%, 4%, 3%, 1% and 0% among the healthy controls, respectively. Healthy controls and boys performed testing at higher ventilation rates (p < .05). Conclusion Eucapnic voluntary hyperventilation testing is feasible among children aged 10-16 years and has diagnostic value in evaluating exercise-induced dyspnoea among children. A minimum 10% fall in FEV1 is a good diagnostic cut-off value. Disease status appears to be important covariates

PCNA ubiquitylation ensures timely completion of unperturbed DNA replication in fission yeast

PCNA ubiquitylation on lysine 164 is required for DNA damage tolerance. In many organisms PCNA is also ubiquitylated in unchallenged S phase but the significance of this has not been established. Using Schizosaccharomyces pombe, we demonstrate that lysine 164 ubiquitylation of PCNA contributes to efficient DNA replication in the absence of DNA damage. Loss of PCNA ubiquitylation manifests most strongly at late replicating regions and increases the frequency of replication gaps. We show that PCNA ubiquitylation increases the proportion of chromatin associated PCNA and the co-immunoprecipitation of Polymerase δ with PCNA during unperturbed replication and propose that ubiquitylation acts to prolong the chromatin association of these replication proteins to allow the efficient completion of Okazaki fragment synthesis by mediating gap filling

Polymerase δ replicates both strands after homologous recombination-dependent fork restart

To maintain genetic stability DNA must be replicated only once and replication completed even when individual replication forks are inactivated. Because fork inactivation is common, the passive convergence of an adjacent fork is insufficient to rescue all inactive forks. Thus, eukaryotic cells have evolved homologous recombination-dependent mechanisms to restart persistent inactive forks. Completing DNA synthesis via Homologous Recombination Restarted Replication (HoRReR) ensures cell survival, but at a cost. One such cost is increased mutagenesis caused by HoRReR being more error prone than canonical replication. This increased error rate implies that the HoRReR mechanism is distinct from that of a canonical fork. Here we exploit the fission yeast Schizosaccharomyces pombe to demonstrate that a DNA sequence duplicated by HoRReR during S phase is replicated semi-conservatively, but that both the leading and lagging strands are synthesised by DNA polymerase delta

Sensing of Replication Stress and Mec1 Activation Act through Two Independent Pathways Involving the 9-1-1 Complex and DNA Polymerase ε

Following DNA damage or replication stress, budding yeast cells activate the Rad53 checkpoint kinase, promoting genome stability in these challenging conditions. The DNA damage and replication checkpoint pathways are partially overlapping, sharing several factors, but are also differentiated at various levels. The upstream kinase Mec1 is required to activate both signaling cascades together with the 9-1-1 PCNA-like complex and the Dpb11 (hTopBP1) protein. After DNA damage, Dpb11 is also needed to recruit the adaptor protein Rad9 (h53BP1). Here we analyzed the mechanisms leading to Mec1 activation in vivo after DNA damage and replication stress. We found that a ddc1Δdpb11-1 double mutant strain displays a synthetic defect in Rad53 and H2A phosphorylation and is extremely sensitive to hydroxyurea (HU), indicating that Dpb11 and the 9-1-1 complex independently promote Mec1 activation. A similar phenotype is observed when both the 9-1-1 complex and the Dpb4 non-essential subunit of DNA polymerase ε (Polε) are contemporarily absent, indicating that checkpoint activation in response to replication stress is achieved through two independent pathways, requiring the 9-1-1 complex and Polε

SH3 Domain-Mediated Recruitment of Host Cell Amphiphysins by Alphavirus nsP3 Promotes Viral RNA Replication

Among the four non-structural proteins of alphaviruses the function of nsP3 is the least well understood. NsP3 is a component of the viral replication complex, and composed of a conserved aminoterminal macro domain implicated in viral RNA synthesis, and a poorly conserved carboxyterminal region. Despite the lack of overall homology we noted a carboxyterminal proline-rich sequence motif shared by many alphaviral nsP3 proteins, and found it to serve as a preferred target site for the Src-homology 3 (SH3) domains of amphiphysin-1 and -2. Nsp3 proteins of Semliki Forest (SFV), Sindbis (SINV), and Chikungunya viruses all showed avid and SH3-dependent binding to amphiphysins. Upon alphavirus infection the intracellular distribution of amphiphysin was dramatically altered and colocalized with nsP3. Mutations in nsP3 disrupting the amphiphysin SH3 binding motif as well as RNAi-mediated silencing of amphiphysin-2 expression resulted in impaired viral RNA replication in HeLa cells infected with SINV or SFV. Infection of Balb/c mice with SFV carrying an SH3 binding-defective nsP3 was associated with significantly decreased mortality. These data establish SH3 domain-mediated binding of nsP3 with amphiphysin as an important host cell interaction promoting alphavirus replication

Characterization of Human DNA Polymerase Delta and Its Subassemblies Reconstituted by Expression in the Multibac System

Mammalian DNA polymerase δ (Pol δ), a four-subunit enzyme, plays a crucial and versatile role in DNA replication and DNA repair processes. We have reconstituted human Pol δ complexes in insect cells infected with a single baculovirus into which one or more subunits were assembled. This system allowed for the efficient expression of the tetrameric Pol δ holoenzyme, the p125/p50 core dimer, the core+p68 trimer and the core+p12 trimer, as well as the p125 catalytic subunit. These were isolated in milligram amounts with reproducible purity and specific activities by a highly standardized protocol. We have systematically compared their activities in order to gain insights into the roles of the p12 and p68 subunits, as well as their responses to PCNA. The relative specific activities (apparent kcat) of the Pol δ holoenzyme, core+p68, core+p12 and p125/p50 core were 100, 109, 40, and 29. The corresponding apparent Kd's for PCNA were 7.1, 8.7, 9.3 and 73 nM. Our results support the hypothesis that Pol δ interacts with PCNA through multiple interactions, and that there may be a redundancy in binding interactions that may permit Pol δ to adopt flexible configurations with PCNA. The abilities of the Pol δ complexes to fully extend singly primed M13 DNA were examined. All the subassemblies except the core+p68 were defective in their abilities to completely extend the primer, showing that the p68 subunit has an important function in synthesis of long stretches of DNA in this assay. The core+p68 trimer could be reconstituted by addition of p12

The Major Roles of DNA Polymerases Epsilon and Delta at the Eukaryotic Replication Fork Are Evolutionarily Conserved

Coordinated replication of eukaryotic genomes is intrinsically asymmetric, with continuous leading strand synthesis preceding discontinuous lagging strand synthesis. Here we provide two types of evidence indicating that, in fission yeast, these two biosynthetic tasks are performed by two different replicases. First, in Schizosaccharomyces pombe strains encoding a polδ-L591M mutator allele, base substitutions in reporter genes placed in opposite orientations relative to a well-characterized replication origin are strand-specific and distributed in patterns implying that Polδ is primarily involved in lagging strand replication. Second, in strains encoding a polε-M630F allele and lacking the ability to repair rNMPs in DNA due to a defect in RNase H2, rNMPs are selectively observed in nascent leading strand DNA. The latter observation demonstrates that abundant rNMP incorporation during replication can be tolerated and that they are normally removed in an RNase H2-dependent manner. This provides strong physical evidence that Polε is the primary leading strand replicase. Collectively, these data and earlier results in budding yeast indicate that the major roles of Polδ and Polε at the eukaryotic replication fork are evolutionarily conserved