Electron microscopic visualization of tRNA genes with ferritin-avidin: biotin labels

A method is described for indirect electron microscopic visualization and mapping of tRNA and other short transcripts hybridized to DNA. This method depends upon the attachment of the electron-dense protein ferritin to the RNA, the binding being mediated by the remarkably strong association of the egg white protein avidin with biotin. Biotin is covalently attached to the 3' end of tRNA using an NH2 (CH2) 5NH2 bridge. The tRNA-biotin adduct is hybridized to complementcrry DNA sequences present in a single stranded nonhomology loop of a DNA:DNA heteroduplex. Avidin, covalently crosslinked to ferritin is mixed with the heteroduplex and becomes bound to the hybridized tRNA-biotin. Observation of the DNA:RNA-biotin:avidin-ferritin complex by electron microsdopy specifically and accurately reveals the position of the tRNA gene, with a frequency of labeling of approximately 50%

PIK3CA-mediated PI3-kinase signalling is essential for HPV-induced transformation in vitro

<p>Abstract</p> <p>Background</p> <p>High-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development. The additional (epi)genetic alterations driving malignant transformation of hrHPV-infected cells however, are not yet fully elucidated. In this study we experimentally assessed the role of the PI3-kinase pathway and its regulator PIK3CA, which is frequently altered in cervical cancer, in HPV-induced transformation.</p> <p>Methods</p> <p>Cervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal <it>in vitro </it>model system of hrHPV-transfected keratinocytes, representing the immortal and anchorage independent phenotype, was assayed for PI3-kinase activation and function using chemical pathway inhibition i.e. LY294002 treatment, and PIK3CA RNA interference. Phenotypes examined included cellular viability, migration, anchorage independent growth and differentiation. mRNA expression of hTERT and HPV16 E6E7 were studied using quantitative RT-PCR and Northern blotting.</p> <p>Results</p> <p>Cervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPV-induced transformation <it>in vitro</it>, expression of the catalytic subunit PIK3CA as well as activation of downstream effector PKB/AKT progressively increased in parallel. Inhibition of PI3-kinase signalling in HPV16-transfected keratinocytes by chemical interference or siRNA-mediated silencing of PIK3CA resulted in a decreased phosphorylation of PKB/AKT. Moreover, blockage of PI3-kinase resulted in reduced cellular viability, migration, and anchorage independent growth. These properties were accompanied with a downregulation of HPV16E7 and hTERT mRNA expression. In organotypic raft cultures of HPV16- and HPV18-immortalized cells, phosphorylated PKB/AKT was primarily seen in differentiated cells staining positive for cytokeratin 10 (CK10). Upon PI3-kinase signalling inhibition, there was a severe impairment in epithelial tissue development as well as a dramatic reduction in p-PKB/AKT and CK10.</p> <p>Conclusion</p> <p>The present data indicate that activation of the PI3-kinase/PKB/AKT pathway through PIK3CA regulates various transformed phenotypes as well as growth and differentiation of HPV-immortalized cells and may therefore play a pivotal role in HPV-induced carcinogenesis.</p

Remodeling of the Human Papillomavirus Type 11 Replication Origin into Discrete Nucleoprotein Particles and Looped Structures by the E2 Protein

The human papillomavirus (HPV) origin (ori) of DNA replication shares a common theme with many DNA control elements in having multiple binding sites for one or more proteins spaced over several hundred base pairs. The HPV type-11 ori spans 103 bp and contains three palindromic binding sites (E2BS-2, E2BS-3, and E2BS-4) for the dimeric E2 origin binding protein. These sites are separated by 64 bp and 3 bp. E2BS-1 is located 288 bp upstream of E2BS-2 and is not required for efficient transient or cell-free replication. In this study, electron microscopy was used to visualize complexes of HPV-11 ori DNA bound by purified E2 protein. DNA containing only E2BS-2 showed a single E2 dimer bound. DNA containing E2BS-3 and E2BS-4 showed two side-by-side E2 dimers, while DNA containing E2BS-2, E2BS-3, and E2BS-4 exhibited a large disk/ring-shaped protein particle bound indicating that the DNA had been remodeled into a discrete complex, likely containing an E2 hexamer. With all four binding sites present, up to 27% of the DNA molecules were arranged into loops by E2, the majority of which spanned E2BS-1 and one of the other three sites. Studies of the dependence of looping on salt, ATP, and DTT using full length E2 and an E2 protein containing only the carboxyl-terminal DNA binding and protein dimerization domain suggest that looping is dependent on the N terminal domain as well as factors which may affect the manner in which E2 scans DNA for binding sites. The role of these structures in the modeling and regulation of the HPV-11 ori is discussed

Human Hsp70 and Hsp40 Chaperone Proteins Facilitate Human Papillomavirus-11 E1 Protein Binding to the Origin and Stimulate Cell-free DNA Replication

Human papillomavirus replication initiator, the E1 helicase, binds weakly to the origin of DNA replication. Purified human chaperone proteins Hsp70 and Hsp40 (HDJ-1 and HDJ-2) independently and additively enhanced E1 binding to the origin. The interaction between E1 and Hsp70 was transient and required ATP hydrolysis, whereas Hsp40 bound to E1 directly and remained in the complex. A peptide of 20 residues spanning the HPD loop and helix II of the J domain of YDJ-1 also stimulated E1 binding to the origin, alone or in combination with Hsp70 or Hsp40. A mutated peptide (H34Q) had a reduced activity, while an adjacent or an overlapping peptide had no effect. Neither Hsp70 nor the J peptide altered the E1/DNA ratio in the complex. Electron microscopy showed that E1 mainly bound to DNA as a hexamer. In the presence of Hsp40, E1 primarily bound to DNA as a dihexamer. Preincubation of chaperones with viral E1 and template shortened the lag time and increased replication in a cell-free system. Since two helicases are essential for bidirectional replication of human papillomavirus DNA, these results demonstrate that, as in prokaryotes, chaperones play an important role in the assembly of preinitiation complexes on the origin

Multi-particle azimuthal correlations in p-Pb and Pb-Pb collisions at the CERN Large Hadron Collider

Measurements of multi-particle azimuthal correlations (cumulants) for charged particles in p-Pb and Pb-Pb collisions are presented. They help address the question of whether there is evidence for global, flow-like, azimuthal correlations in the p-Pb system. Comparisons are made to measurements from the larger Pb-Pb system, where such evidence is established. In particular, the second harmonic two-particle cumulants are found to decrease with multiplicity, characteristic of a dominance of few-particle correlations in p-Pb collisions. However, when a $|\Delta \eta|$ gap is placed to suppress such correlations, the two-particle cumulants begin to rise at high-multiplicity, indicating the presence of global azimuthal correlations. The Pb-Pb values are higher than the p-Pb values at similar multiplicities. In both systems, the second harmonic four-particle cumulants exhibit a transition from positive to negative values when the multiplicity increases. The negative values allow for a measurement of $v_{2}\{4\}$ to be made, which is found to be higher in Pb-Pb collisions at similar multiplicities. The second harmonic six-particle cumulants are also found to be higher in Pb-Pb collisions. In Pb-Pb collisions, we generally find $v_{2}\{4\} \simeq v_{2}\{6\}\neq 0$ which is indicative of a Bessel-Gaussian function for the $v_{2}$ distribution. For very high-multiplicity Pb-Pb collisions, we observe that the four- and six-particle cumulants become consistent with 0. Finally, third harmonic two-particle cumulants in p-Pb and Pb-Pb are measured. These are found to be similar for overlapping multiplicities, when a $|\Delta\eta| > 1.4$ gap is placed.Comment: 25 pages, 11 captioned figures, 3 tables, authors from page 20, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/87

Multiplicity dependence of jet-like two-particle correlations in p-Pb collisions at sNN\sqrt{s_{NN}} = 5.02 TeV

Two-particle angular correlations between unidentified charged trigger and associated particles are measured by the ALICE detector in p-Pb collisions at a nucleon-nucleon centre-of-mass energy of 5.02 TeV. The transverse-momentum range 0.7 $< p_{\rm{T}, assoc} < p_{\rm{T}, trig} <$ 5.0 GeV/$c$ is examined, to include correlations induced by jets originating from low momen\-tum-transfer scatterings (minijets). The correlations expressed as associated yield per trigger particle are obtained in the pseudorapidity range $|\eta|<0.9$. The near-side long-range pseudorapidity correlations observed in high-multiplicity p-Pb collisions are subtracted from both near-side short-range and away-side correlations in order to remove the non-jet-like components. The yields in the jet-like peaks are found to be invariant with event multiplicity with the exception of events with low multiplicity. This invariance is consistent with the particles being produced via the incoherent fragmentation of multiple parton--parton scatterings, while the yield related to the previously observed ridge structures is not jet-related. The number of uncorrelated sources of particle production is found to increase linearly with multiplicity, suggesting no saturation of the number of multi-parton interactions even in the highest multiplicity p-Pb collisions. Further, the number scales in the intermediate multiplicity region with the number of binary nucleon-nucleon collisions estimated with a Glauber Monte-Carlo simulation.Comment: 23 pages, 6 captioned figures, 1 table, authors from page 17, published version, figures at http://aliceinfo.cern.ch/ArtSubmission/node/161

Comprehensive Control of Human Papillomavirus Infections and Related Diseases

Infection with human papillomavirus (HPV) is recognized as one of the major causes of infection-related cancer worldwide, as well as the causal factor in other diseases. Strong evidence for a causal etiology with HPV has been stated by the International Agency for Research on Cancer for cancers of the cervix uteri, penis, vulva, vagina, anus and oropharynx (including base of the tongue and tonsils). Of the estimated 12.7 million new cancers occurring in 2008 worldwide, 4.8% were attributable to HPV infection, with substantially higher incidence and mortality rates seen in developing versus developed countries. In recent years, we have gained tremendous knowledge about HPVs and their interactions with host cells, tissues and the immune system; have validated and implemented strategies for safe and efficacious prophylactic vaccination against HPV infections; have developed increasingly sensitive and specific molecular diagnostic tools for HPV detection for use in cervical cancer screening; and have substantially increased global awareness of HPV and its many associated diseases in women, men, and children. While these achievements exemplify the success of biomedical research in generating important public health interventions, they also generate new and daunting challenges: costs of HPV prevention and medical care, the implementation of what is technically possible, socio-political resistance to prevention opportunities, and the very wide ranges of national economic capabilities and health care systems. Gains and challenges faced in the quest for comprehensive control of HPV infection and HPV-related cancers and other disease are summarized in this review. The information presented may be viewed in terms of a reframed paradigm of prevention of cervical cancer and other HPV-related diseases that will include strategic combinations of at least four major components: 1) routine introduction of HPV vaccines to women in all countries, 2) extension and simplification of existing screening programs using HPV-based technology, 3) extension of adapted screening programs to developing populations, and 4) consideration of the broader spectrum of cancers and other diseases preventable by HPV vaccination in women, as well as in men. Despite the huge advances already achieved, there must be ongoing efforts including international advocacy to achieve widespread optimally universal implementation of HPV prevention strategies in both developed and developing countries. This article summarizes information from the chapters presented in a special ICO Monograph 'Comprehensive Control of HPV Infections and Related Diseases' Vaccine Volume 30, Supplement 5, 2012. Additional details on each subtopic and full information regarding the supporting literature references may be found in the original chapters. (C) 2013 Elsevier Ltd. All rights reserved

K0S and Λ production in Pb-Pb collisions at sNN−−−−√=2.76 TeV

The ALICE measurement of K0S and Λ production at midrapidity in Pb-Pb collisions at sNN−−−√=2.76  TeV is presented. The transverse momentum (pT) spectra are shown for several collision centrality intervals and in the pT range from 0.4  GeV/c (0.6  GeV/c for Λ) to 12  GeV/c. The pT dependence of the Λ/K0S ratios exhibits maxima in the vicinity of 3  GeV/c, and the positions of the maxima shift towards higher pT with increasing collision centrality. The magnitude of these maxima increases by almost a factor of three between most peripheral and most central Pb-Pb collisions. This baryon excess at intermediate pT is not observed in pp interactions at s√=0.9  TeV and at s√=7  TeV. Qualitatively, the baryon enhancement in heavy-ion collisions is expected from radial flow. However, the measured pT spectra above 2  GeV/c progressively decouple from hydrodynamical-model calculations. For higher values of pT, models that incorporate the influence of the medium on the fragmentation and hadronization processes describe qualitatively the pT dependence of the Λ/K0S ratio

Enhanced production of multi-strange hadrons in high-multiplicity proton-proton collisions

At sufficiently high temperature and energy density, nuclear matter undergoes a transition to a phase in which quarks and gluons are not confined: the quark-gluon plasma (QGP)(1). Such an exotic state of strongly interacting quantum chromodynamics matter is produced in the laboratory in heavy nuclei high-energy collisions, where an enhanced production of strange hadrons is observed(2-6). Strangeness enhancement, originally proposed as a signature of QGP formation in nuclear collisions(7), is more pronounced for multi-strange baryons. Several effects typical of heavy-ion phenomenology have been observed in high-multiplicity proton-proton (pp) collisions(8,9), but the enhanced production of multi-strange particles has not been reported so far. Here we present the first observation of strangeness enhancement in high-multiplicity proton-proton collisions. We find that the integrated yields of strange and multi-strange particles, relative to pions, increases significantly with the event charged-particle multiplicity. The measurements are in remarkable agreement with the p-Pb collision results(10,11), indicating that the phenomenon is related to the final system created in the collision. In high-multiplicity events strangeness production reaches values similar to those observed in Pb-Pb collisions, where a QGP is formed.Peer reviewe