Actin binding domains direct actin-binding proteins to different cytoskeletal locations

<p>Abstract</p> <p>Background</p> <p>Filamin (FLN) and non-muscle α-actinin are members of a family of F-actin cross-linking proteins that utilize Calponin Homology domains (CH-domain) for actin binding. Although these two proteins have been extensively characterized, little is known about what regulates their binding to F-actin filaments in the cell.</p> <p>Results</p> <p>We have constructed fusion proteins consisting of green fluorescent protein (GFP) with either the entire cross-linking protein or its actin-binding domain (ABD) and examined the localization of these fluorescent proteins in living cells under a variety of conditions. The full-length fusion proteins, but not the ABD's complemented the defects of cells lacking both endogenous proteins indicating that they are functional. The localization patterns of filamin (GFP-FLN) and α-actinin (GFP-αA) were overlapping but distinct. GFP-FLN localized to the peripheral cell cortex as well as to new pseudopods of unpolarized cells, but was observed to localize to the rear of polarized cells during cAMP and folate chemotaxis. GFP-αA was enriched in new pseudopods and at the front of polarized cells, but in all cases was absent from the peripheral cortex. Although both proteins appear to be involved in macropinocytosis, the association time of the GFP-probes with the internalized macropinosome differed. Surprisingly, the localization of the GFP-actin-binding domain fusion proteins precisely reflected that of their respective full length constructs, indicating that the localization of the protein was determined by the actin-binding domain alone. When expressed in a cell line lacking both filamin and α-actinin, the probes maintain their distinct localization patterns suggesting that they are not functionally redundant.</p> <p>Conclusion</p> <p>These observations strongly suggest that the regulation of the binding of these proteins to actin filaments is built into the actin-binding domains. We suggest that different actin binding domains have different affinities for F-actin filaments in functionally distinct regions of the cytoskeleton.</p

Do I Have My Attention? Speed of Processing Advantages for the Self-Face Are Not Driven by Automatic Attention Capture

We respond more quickly to our own face than to other faces, but there is debate over whether this is connected to attention-grabbing properties of the self-face. In two experiments, we investigate whether the self-face selectively captures attention, and the attentional conditions under which this might occur. In both experiments, we examined whether different types of face (self, friend, stranger) provide differential levels of distraction when processing self, friend and stranger names. In Experiment 1, an image of a distractor face appeared centrally – inside the focus of attention – behind a target name, with the faces either upright or inverted. In Experiment 2, distractor faces appeared peripherally – outside the focus of attention – in the left or right visual field, or bilaterally. In both experiments, self-name recognition was faster than other name recognition, suggesting a self-referential processing advantage. The presence of the self-face did not cause more distraction in the naming task compared to other types of face, either when presented inside (Experiment 1) or outside (Experiment 2) the focus of attention. Distractor faces had different effects across the two experiments: when presented inside the focus of attention (Experiment 1), self and friend images facilitated self and friend naming, respectively. This was not true for stranger stimuli, suggesting that faces must be robustly represented to facilitate name recognition. When presented outside the focus of attention (Experiment 2), no facilitation occurred. Instead, we report an interesting distraction effect caused by friend faces when processing strangers’ names. We interpret this as a “social importance” effect, whereby we may be tuned to pick out and pay attention to familiar friend faces in a crowd. We conclude that any speed of processing advantages observed in the self-face processing literature are not driven by automatic attention capture

Genetic Diversity of EBV-Encoded LMP1 in the Swiss HIV Cohort Study and Implication for NF-Κb Activation

Epstein-Barr virus (EBV) is associated with several types of cancers including Hodgkin's lymphoma (HL) and nasopharyngeal carcinoma (NPC). EBV-encoded latent membrane protein 1 (LMP1), a multifunctional oncoprotein, is a powerful activator of the transcription factor NF-κB, a property that is essential for EBV-transformed lymphoblastoid cell survival. Previous studies reported LMP1 sequence variations and induction of higher NF-κB activation levels compared to the prototype B95-8 LMP1 by some variants. Here we used biopsies of EBV-associated cancers and blood of individuals included in the Swiss HIV Cohort Study (SHCS) to analyze LMP1 genetic diversity and impact of sequence variations on LMP1-mediated NF-κB activation potential. We found that a number of variants mediate higher NF-κB activation levels when compared to B95-8 LMP1 and mapped three single polymorphisms responsible for this phenotype: F106Y, I124V and F144I. F106Y was present in all LMP1 isolated in this study and its effect was variant dependent, suggesting that it was modulated by other polymorphisms. The two polymorphisms I124V and F144I were present in distinct phylogenetic groups and were linked with other specific polymorphisms nearby, I152L and D150A/L151I, respectively. The two sets of polymorphisms, I124V/I152L and F144I/D150A/L151I, which were markers of increased NF-κB activation in vitro, were not associated with EBV-associated HL in the SHCS. Taken together these results highlighted the importance of single polymorphisms for the modulation of LMP1 signaling activity and demonstrated that several groups of LMP1 variants, through distinct mutational paths, mediated enhanced NF-κB activation levels compared to B95-8 LMP1

Sequence analysis of the Epstein-Barr virus (EBV) BRLF1 gene in nasopharyngeal and gastric carcinomas

<p>Abstract</p> <p>Background</p> <p>Epstein-Barr virus (EBV) has a biphasic infection cycle consisting of a latent and a lytic replicative phase. The product of immediate-early gene BRLF1, Rta, is able to disrupt the latency phase in epithelial cells and certain B-cell lines. The protein Rta is a frequent target of the EBV-induced cytotoxic T cell response. In spite of our good understanding of this protein, little is known for the gene polymorphism of BRLF1.</p> <p>Results</p> <p>BRLF1 gene was successfully amplified in 34 EBV-associated gastric carcinomas (EBVaGCs), 57 nasopharyngeal carcinomas (NPCs) and 28 throat washings (TWs) samples from healthy donors followed by PCR-direct sequencing. Fourteen loci were found to be affected by amino acid changes, 17 loci by silent nucleotide changes. According to the phylogenetic tree, 5 distinct subtypes of BRLF1 were identified, and 2 subtypes BR1-A and BR1-C were detected in 42.9% (51/119), 42.0% (50/119) of samples, respectively. The distribution of these 2 subtypes among 3 types of specimens was significantly different. The subtype BR1-A preferentially existed in healthy donors, while BR1-C was seen more in biopsies of NPC. A silent mutation A/G was detected in all the isolates. Among 3 functional domains, the dimerization domain of Rta showed a stably conserved sequence, while DNA binding and transactivation domains were detected to have multiple mutations. Three of 16 CTL epitopes, NAA, QKE and ERP, were affected by amino acid changes. Epitope ERP was relatively conserved; epitopes NAA and QKE harbored more mutations.</p> <p>Conclusions</p> <p>This first detailed investigation of sequence variations in BRLF1 gene has identified 5 distinct subtypes. Two subtypes BR1-A and BR1-C are the dominant genotypes of BRLF1. The subtype BR1-C is more frequent in NPCs, while BR1-A preferentially presents in healthy donors. BR1-C may be associated with the tumorigenesis of NPC.</p

Respiratory Syncytial Virus NS1 Protein Colocalizes with Mitochondrial Antiviral Signaling Protein MAVS following Infection

Respiratory syncytial virus (RSV) nonstructural protein 1(NS1) attenuates type-I interferon (IFN) production during RSV infection; however the precise role of RSV NS1 protein in orchestrating the early host-virus interaction during infection is poorly understood. Since NS1 constitutes the first RSV gene transcribed and the production of IFN depends upon RLR (RIG-I-like receptor) signaling, we reasoned that NS1 may interfere with this signaling. Herein, we report that NS1 is localized to mitochondria and binds to mitochondrial antiviral signaling protein (MAVS). Live-cell imaging of rgRSV-infected A549 human epithelial cells showed that RSV replication and transcription occurs in proximity to mitochondria. NS1 localization to mitochondria was directly visualized by confocal microscopy using a cell-permeable chemical probe for His6-NS1. Further, NS1 colocalization with MAVS in A549 cells infected with RSV was shown by confocal laser microscopy and immuno-electron microscopy. NS1 protein is present in the mitochondrial fraction and co-immunoprecipitates with MAVS in total cell lysatesof A549 cells transfected with the plasmid pNS1-Flag. By immunoprecipitation with anti-RIG-I antibody, RSV NS1 was shown to associate with MAVS at an early stage of RSV infection, and to disrupt MAVS interaction with RIG-I (retinoic acid inducible gene) and the downstream IFN antiviral and inflammatory response. Together, these results demonstrate that NS1 binds to MAVS and that this binding inhibits the MAVS-RIG-I interaction required for IFN production

Jet energy measurement with the ATLAS detector in proton-proton collisions at root s=7 TeV

The jet energy scale and its systematic uncertainty are determined for jets measured with the ATLAS detector at the LHC in proton-proton collision data at a centre-of-mass energy of √s = 7TeV corresponding to an integrated luminosity of 38 pb-1. Jets are reconstructed with the anti-kt algorithm with distance parameters R=0. 4 or R=0. 6. Jet energy and angle corrections are determined from Monte Carlo simulations to calibrate jets with transverse momenta pT≥20 GeV and pseudorapidities {pipe}η{pipe}<4. 5. The jet energy systematic uncertainty is estimated using the single isolated hadron response measured in situ and in test-beams, exploiting the transverse momentum balance between central and forward jets in events with dijet topologies and studying systematic variations in Monte Carlo simulations. The jet energy uncertainty is less than 2. 5 % in the central calorimeter region ({pipe}η{pipe}<0. 8) for jets with 60≤pT<800 GeV, and is maximally 14 % for pT<30 GeV in the most forward region 3. 2≤{pipe}η{pipe}<4. 5. The jet energy is validated for jet transverse momenta up to 1 TeV to the level of a few percent using several in situ techniques by comparing a well-known reference such as the recoiling photon pT, the sum of the transverse momenta of tracks associated to the jet, or a system of low-pT jets recoiling against a high-pT jet. More sophisticated jet calibration schemes are presented based on calorimeter cell energy density weighting or hadronic properties of jets, aiming for an improved jet energy resolution and a reduced flavour dependence of the jet response. The systematic uncertainty of the jet energy determined from a combination of in situ techniques is consistent with the one derived from single hadron response measurements over a wide kinematic range. The nominal corrections and uncertainties are derived for isolated jets in an inclusive sample of high-pT jets. Special cases such as event topologies with close-by jets, or selections of samples with an enhanced content of jets originating from light quarks, heavy quarks or gluons are also discussed and the corresponding uncertainties are determined. © 2013 CERN for the benefit of the ATLAS collaboration

Two Major Medicinal Honeys Have Different Mechanisms of Bactericidal Activity

Honey is increasingly valued for its antibacterial activity, but knowledge regarding the mechanism of action is still incomplete. We assessed the bactericidal activity and mechanism of action of Revamil® source (RS) honey and manuka honey, the sources of two major medical-grade honeys. RS honey killed Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa within 2 hours, whereas manuka honey had such rapid activity only against B. subtilis. After 24 hours of incubation, both honeys killed all tested bacteria, including methicillin-resistant Staphylococcus aureus, but manuka honey retained activity up to higher dilutions than RS honey. Bee defensin-1 and H2O2 were the major factors involved in rapid bactericidal activity of RS honey. These factors were absent in manuka honey, but this honey contained 44-fold higher concentrations of methylglyoxal than RS honey. Methylglyoxal was a major bactericidal factor in manuka honey, but after neutralization of this compound manuka honey retained bactericidal activity due to several unknown factors. RS and manuka honey have highly distinct compositions of bactericidal factors, resulting in large differences in bactericidal activity

Epstein-Barr Virus latent membrane protein 1 induces Snail and epithelial–mesenchymal transition in metastatic nasopharyngeal carcinoma

Background:Epstein-Barr Virus (EBV)-associated nasopharyngeal carcinoma (NPC) is distinctive among head-and-neck cancers in its undifferentiated histopathology and highly metastatic character. We have recently investigated the involvement of epithelial–mesenchymal transition (EMT) in NPC. In a previous study, we found a close association of expression of LMP1, the principal EBV oncoprotein, with expression of Twist and induction of EMT.Methods:We analysed expression of Snail in 41 NPC tissues by immunohistochemistry. The role of Twist as well as Snail in EMT of NPC was investigated by using NP69SV40T human nasopharyngeal cells.Results:In NPC tissues, overexpression of Snail is associated with expression of LMP1 in carcinomatous cells. In addition, expression of Snail positively correlated with metastasis and independently correlated inversely with expression of E-cadherin. Expression of Twist had no association with expression of E-cadherin. Further, in a human nasopharyngeal cell line, LMP1 induces EMT and its associated cellular motility and invasiveness. Expression of Snail is induced by LMP1 in these cells, and small hairpin RNA (shRNA) to Snail reversed the cellular changes. By contrast, Twist did not produce EMT in these nasopharyngeal cells.Conclusions:This study strengthens the association of EMT with the metastatic behaviour of NPC. These results suggest that induction of Snail by the EBV oncoprotein LMP1 has a pivotal role in EMT in NPC