Adenovirus E1A directly targets the E2F/DP-1 complex

Deregulation of the cell cycle is of paramount importance during adenovirus infection. Adenovirus normally infects quiescent cells and must initiate the cell cycle in order to propagate itself. The pRb family of proteins controls entry into the cell cycle by interacting with and repressing transcriptional activation by the E2F transcription factors. The viral E1A proteins indirectly activate E2F-dependent transcription and cell cycle entry, in part, by interacting with pRb and family members to free the E2Fs. We report here that an E1A 13S isoform can unexpectedly activate E2F-responsive gene expression independently of binding to the pRb family of proteins. We demonstrate that E1A binds to E2F/DP-1 complexes through a direct interaction with DP-1. E1A appears to utilize this binding to recruit itself to E2F-regulated promoters, and this allows the E1A 13S protein, but not the E1A 12S protein, to activate transcription independently of interaction with pRb. Importantly, expression of E1A 13S, but not E1A 12S, led to significant enhancement of E2F4 occupancy of E2F sites of two E2F-regulated promoters. These observations identify a novel mechanism by which adenovirus deregulates the cell cycle and suggest that E1A 13S may selectively activate a subset of E2F-regulated cellular genes during infection. © 2011, American Society for Microbiology

An Assist for Cognitive Diagnostics in Soccer: Two Valid Tasks Measuring Inhibition and Cognitive Flexibility in a Soccer-Specific Setting With a Soccer-Specific Motor Response

In professional soccer, players, coaches, and researchers alike recognize the importance of cognitive skills. Research addressing the relevance of cognitive skills has been based on the cognitive component skills approach (i.e., general cognitive processes) or the expert performance approach (i.e., sport-specific cognitive processes). Our project aimed to combine the strengths of both approaches to develop and validate cognitive tasks measuring inhibition and cognitive flexibility in a soccer-specific setting with a soccer-specific motor response. In the main study 77 elite youth soccer players completed a computerized version of the standard flanker and number–letter tasks as well as flanker and number–letter tasks requiring a soccer-specific motor response (i.e., pass) in a soccer-specific setting (i.e., the SoccerBot360). Results show good reliability for both tasks. For the SoccerBot360 number–letter task, switch effects for response times and accuracy and acceptable convergent validity were shown. A flanker effect for response time but not accuracy was apparent. Due to no acceptable convergent validity, the flanker task was revised (i.e., adaptation of stimuli) and 63 adult soccer players participated in a follow-up validation study in the SoccerBot100. The revised flanker task showed the flanker effect for response time, but not for accuracy. However, acceptable convergent validity for response time was present. Thus, the soccer-specific number–letter and to some extent the soccer-specific flanker task show potential to be used as a valid cognitive diagnostic tool by soccer clubs.Peer Reviewe

Urinary 1-Hydroxypyrene as a Biomarker of PAH Exposure in 3-Year-Old Ukrainian Children

Urinary 1-hydroxypyrene (1-OHP) is a biomarker of polycyclic aromatic hydrocarbon (PAH) exposure. We measured urinary 1-OHP in 48 children 3 years of age in Mariupol, Ukraine, who lived near a steel mill and coking facility and compared these with 1-OHP concentrations measured in 42 children of the same age living in the capital city of Kiev, Ukraine. Children living in Mariupol had significantly higher urinary 1-OHP and creatinine-adjusted urinary 1-OHP than did children living in Kiev (adjusted: 0.69 vs. 0.34 μmol/mol creatinine, p < 0.001; unadjusted: 0.42 vs. 0.30 ng/mL, p = 0.002). Combined, children in both cities exposed to environmental tobacco smoke in their homes had higher 1-OHP than did children not exposed (0.61 vs. 0.42 μmol/mol creatinine; p = 0.04; p = 0.07 after adjusting for city). In addition, no significant differences were seen with sex of the children. Our sample of children in Mariupol has the highest reported mean urinary 1-OHP concentrations in children studied to date, most likely due to their proximity to a large industrial point source of PAHs

Control of human adenovirus type 5 gene expression by cellular Daxx/ATRX chromatin-associated complexes

Death domain–associated protein (Daxx) cooperates with X-linked α-thalassaemia retardation syndrome protein (ATRX), a putative member of the sucrose non-fermentable 2 family of ATP-dependent chromatin-remodelling proteins, acting as the core ATPase subunit in this complex, whereas Daxx is the targeting factor, leading to histone deacetylase recruitment, H3.3 deposition and transcriptional repression of cellular promoters. Despite recent findings on the fundamental importance of chromatin modification in host-cell gene regulation, it remains unclear whether adenovirus type 5 (Ad5) transcription is regulated by cellular chromatin remodelling to allow efficient virus gene expression. Here, we focus on the repressive role of the Daxx/ATRX complex during Ad5 replication, which depends on intact protein–protein interaction, as negative regulation could be relieved with a Daxx mutant that is unable to interact with ATRX. To ensure efficient viral replication, Ad5 E1B-55K protein inhibits Daxx and targets ATRX for proteasomal degradation in cooperation with early region 4 open reading frame protein 6 and cellular components of a cullin-dependent E3-ubiquitin ligase. Our studies illustrate the importance and diversity of viral factors antagonizing Daxx/ATRX-mediated repression of viral gene expression and shed new light on the modulation of cellular chromatin remodelling factors by Ad5. We show for the first time that cellular Daxx/ATRX chromatin remodelling complexes play essential roles in Ad gene expression and illustrate the importance of early viral proteins to counteract cellular chromatin remodelling

Transcriptional control by adenovirus E1A conserved region 3 via p300/CBP

The human adenovirus type 5 (HAdV-5) E1A 13S oncoprotein is a potent regulator of gene expression and is used extensively as a model for transcriptional activation. It possesses two independent transcriptional activation domains located in the N-terminus/conserved region (CR) 1 and CR3. The protein acetyltransferase p300 was previously identified by its association with the N-terminus/CR1 portion of E1A and this association is required for oncogenic transformation by E1A. We report here that transcriptional activation by 13S E1A is inhibited by co-expression of sub-stoichiometric amounts of the smaller 12S E1A isoform, which lacks CR3. Transcriptional inhibition by E1A 12S maps to the N-terminus and correlates with the ability to bind p300/CBP, suggesting that E1A 12S is sequestering this limiting factor from 13S E1A. This is supported by the observation that the repressive effect of E1A 12S is reversed by expression of exogenous p300 or CBP, but not by a CBP mutant lacking actyltransferase activity. Furthermore, we show that transcriptional activation by 13S E1A is greatly reduced by siRNA knockdown of p300 and that CR3 binds p300 independently of the well-characterized N-terminal/CR1-binding site. Importantly, CR3 is also required to recruit p300 to the adenovirus E4 promoter during infection. These results identify a new functionally significant interaction between E1A CR3 and the p300/CBP acetyltransferases, expanding our understanding of the mechanism by which this potent transcriptional activator functions

Visualizing Ultrafast Kinetic Instabilities in Laser-Driven Solids using X-ray Scattering

Ultra-intense lasers that ionize and accelerate electrons in solids to near the speed of light can lead to kinetic instabilities that alter the laser absorption and subsequent electron transport, isochoric heating, and ion acceleration. These instabilities can be difficult to characterize, but a novel approach using X-ray scattering at keV energies allows for their visualization with femtosecond temporal resolution on the few nanometer mesoscale. Our experiments on laser-driven flat silicon membranes show the development of structure with a dominant scale of ~60\unit{nm} in the plane of the laser axis and laser polarization, and ~95\unit{nm} in the vertical direction with a growth rate faster than $0.1/\mathrm{fs}$. Combining the XFEL experiments with simulations provides a complete picture of the structural evolution of ultra-fast laser-induced instability development, indicating the excitation of surface plasmons and the growth of a new type of filamentation instability. These findings provide new insight into the ultra-fast instability processes in solids under extreme conditions at the nanometer level with important implications for inertial confinement fusion and laboratory astrophysics

Transcriptional tools: Small molecules for modulating CBP KIX-dependent transcriptional activators

Previously it was demonstrated that amphipathic isoxazolidines are able to functionally replace the transcriptional activation domains of endogenous transcriptional activators. In addition, in vitro binding studies suggested that a key binding partner of these molecules is the CREB Binding Protein (CBP), more specifically the KIX domain within this protein. Here we show that CBP plays an essential role in the ability of isoxazolidine transcriptional activation domains to activate transcription in cells. Consistent with this model, isoxazolidines are able to function as competitive inhibitors of the activators MLL and Jun, both of which utilize a binding interaction with KIX to up-regulate transcription. Further, modification of the N2 side chain produced three analogs with enhanced potency against Jun-mediated transcription, although increased cytotoxicity was also observed. Collectively these small KIX-binding molecules will be useful tools for dissecting the role of the KIX domain in a variety of pathological processes. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 17–23, 2011.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78234/1/21548_ftp.pd

Readthrough of nonsense mutations in Rett syndrome: evaluation of novel aminoglycosides and generation of a new mouse model

Thirty-five percent of patients with Rett syndrome carry nonsense mutations in the MECP2 gene. We have recently shown in transfected HeLa cells that readthrough of nonsense mutations in the MECP2 gene can be achieved by treatment with gentamicin and geneticin. This study was performed to test if readthrough can also be achieved in cells endogenously expressing mutant MeCP2 and to evaluate potentially more effective readthrough compounds. A mouse model was generated carrying the R168X mutation in the MECP2 gene. Transfected HeLa cells expressing mutated MeCP2 fusion proteins and mouse ear fibroblasts isolated from the new mouse model were treated with gentamicin and the novel aminoglycosides NB30, NB54, and NB84. The localization of the readthrough product was tested by immunofluorescence. Readthrough of the R168X mutation in mouse ear fibroblasts using gentamicin was detected but at lower level than in HeLa cells. As expected, the readthrough product, full-length Mecp2 protein, was located in the nucleus. NB54 and NB84 induced readthrough more effectively than gentamicin, while NB30 was less effective. Readthrough of nonsense mutations can be achieved not only in transfected HeLa cells but also in fibroblasts of the newly generated Mecp2R168X mouse model. NB54 and NB84 were more effective than gentamicin and are therefore promising candidates for readthrough therapy in Rett syndrome patients

Frontrunners in the race to develop a SARS-CoV-2 vaccine

Numerous studies continue to be published on the COVID-19 pandemic that is being caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Given the rapidly evolving global response to SARS-CoV-2, here we primarily review the leading COVID-19 vaccine strategies that are currently in Phase III clinical trials. Nonreplicating viral vector strategies, inactivated virus, recombinant protein subunit vaccines, and nucleic acid vaccine platforms are all being pursued in an effort to combat the infection. Preclinical and clinal trial results of these efforts are examined as well as the characteristics of each vaccine strategy from the humoral and cellular immune responses they stimulate, effects of any adjuvants used, and the potential risks associated with immunization such as antibody-dependent enhancement. A number of promising advancements have been made toward the development of multiple vaccine candidates. Preliminary data now emerging from phase III clinical trials show encouraging results for the protective efficacy and safety of at least 3 frontrunning candidates. There is hope that one or more will emerge as potent weapons to protect against SARS-CoV-2.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Effects of Adenovirus Type 5 E1A Isoforms on Viral Replication in Arrested Human Cells.

Human adenovirus has evolved to infect and replicate in terminally differentiated human epithelial cells, predominantly those within the airway, the gut, or the eye. To overcome the block to viral DNA replication present in these cells, the virus expresses the Early 1A proteins (E1A). These immediate early proteins drive cells into S-phase and induce expression of all other viral early genes. During infection, several E1A isoforms are expressed with proteins of 289, 243, 217, 171, and 55 residues being present for human adenovirus type 5. Here we examine the contribution that the two largest E1A isoforms make to the viral life cycle in growth-arrested normal human fibroblasts. Viruses that express E1A289R were found to replicate better than those that do not express this isoform. Importantly, induction of several viral genes was delayed in a virus expressing E1A243R, with several viral structural proteins undetectable by western blot. We also highlight the changes in E1A isoforms detected during the course of viral infection. Furthermore, we show that viral DNA replication occurs more efficiently, leading to higher number of viral genomes in cells infected with viruses that express E1A289R. Finally, induction of S-phase specific genes differs between viruses expressing different E1A isoforms, with those having E1A289R leading to, generally, earlier activation of these genes. Overall, we provide an overview of adenovirus replication using modern molecular biology approaches and further insights into the contribution that E1A isoforms make to the life cycle of human adenovirus in arrested human fibroblasts